Compounds and methods for detection of enzymes that remove methyl succinyl groups from epsilon-amino lysine moieties

ABSTRACT

Provided is a compound that comprises the structure: 
     
       
         
         
             
             
         
       
         
         
           
             where SIG is a signaling molecule and R 3  is a formyl, a succinyl, a methyl succinyl, or a myristoyl. Also provided is a kit is provided that comprises the above compound, with instructions for determining the presence of the enzyme. Additionally, a method is provided for determining whether a sample has an enzyme that removes a succinyl, a methyl succinyl, a formyl, or a myristoyl moiety from an ε-amino of a lysine. Also provided is a method of determining whether a molecule inhibits an enzyme that removes a succinyl, a methyl succinyl, a formyl, or a myristoyl moiety from an ε-amino of a lysine.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.16/387,992, filed Apr. 18, 2019, which is a divisional of U.S.application Ser. No. 15/944,336, filed Apr. 3, 2018 (now U.S. Pat. No.10,308,974, issued Jun. 4, 2019), which is a divisional of U.S.application Ser. No. 15/464,966, filed Mar. 21, 2017 (now U.S. Pat. No.9,963,734, issued May 8, 2018), which is a division of U.S. applicationSer. No. 12/930,693, filed Jan. 13, 2011 (now U.S. Pat. No. 9,637,773,issued May 2, 2017), all of which are hereby incorporated by referencein their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 30, 2020, isnamed ENZ-98-D1-D1-D1-CON-Application-SL.txt and is 3,847 bytes in size.

BACKGROUND OF THE INVENTION (1) Field of the Invention

The present application generally relates to reagents for detectingenzymes. More specifically, substrates for detecting various enzymesthat remove modifications of ε-amino moieties are provided.

(2) Description of the Related Art

Most sirtuin enzymes, also known as class III histone deacetylases(class III HDACs), catalyze a reaction which couples deacetylation ofprotein ε-acetyllysine residues to the formation of O-acetyl-ADP-riboseand nicotinamide from NAD⁺ (Imai et al., 2000; Tanner et al., 2000;Tanny and Moazed, 2001). Some sirtuins, notably human sirtuins SIRT4 andSIRT6, catalyze an alternative reaction, the transfer of an ADP-ribosylgroup from NAD⁺ to proteins (Liszt et al., 2005; Haigis et al., 2006),although the physiological relevance of these reactions is in question(Du et al., 2009). Sirtuin homologs are found in all forms of life,including the archaea, the bacteria, and both unicellular andmulticellular eukaryotes (Smith et al., 2000; Blander and Guarente,2004; Buck et al., 2004; Frye, 2000). The founding exemplar of thegroup, Sir2 from baker's yeast (Saccharomyces cerevisiae), was named forits role in gene-silencing (Silent information regulator 2; Rusche etal., 2003). Transcriptional silencing by Sir2 is linked to itsdeacetylation of lysines in the N-terminal tails of the histones inchromatin, hence the classification as a class III HDAC. Lysinedeacetylation by sirtuins, however, extends beyond histones. Targets ofsirtuin regulatory deacetylation include mammalian transcription factorssuch as p53 (Luo et al., 2001; Vaziri et al., 2001; Langley et al.,2002), the cytoskeletal protein tubulin (North et al., 2003), and thebacterial enzyme acetyl-CoA synthetase (Starai et al., 2002; Zhao etal., 2004) and its mammalian homologs (Shimazu et al., 2010).

SIRT5, along with two other mammalian sirtuins, SIRT3 and SIRT4, islocalized to the mitochondria (Michishita et al., 2005; Nakagawa et al.,2009). The human SIRT5 gene is located in a chromosomal region in whichabnormalities are associated with malignancies, suggesting a possibleSIRT5 role in cancer (Mahlknecht et al., 2008). Thus far, the beststudied of SIRT5's possible physiological roles is the deacetylation andenhancement of the activity of the mitochondrial matrix enzyme carbamoylphosphate synthase 1 (CPS1), the rate-limiting enzyme for urea synthesisin the urea cycle (Nakagawa et al., 2009). Increased urea synthesis isrequired for removal of nitrogenous waste (ammonia) during periods ofincreased amino acid catabolism, including calorie restriction, fastingand the consumption of a high protein diet. Under these conditions,SIRT5 deacetylation of CPS1 is increased, along with CPS1 activity(Nakagawa et al., 2009). At least in the instance of starvation, theincreased SIRT5 activity may be attributed to increased levels of thesirtuin co-substrate NAD⁺ in the mitochondria, which in turn is due toinduction of the NAD⁺ synthetic pathway enzyme nicotinamidephosphoribosyltransferase, (Nampt) (Nakagawa et al., 2009). It should benoted, however, that two proteomic studies of the mouse mitochondrial“acetylome” are in possible conflict with the CPS1 results of Nakagawaet al. (2009). One group observed that calorie restriction increasedacetylation at 7 of 24 sites in CPS1, but did not lead to deacetylationat any sites (Schwer et al., 2009). A comparison of the acetylatedproteins of fed and fasted mice found that fasting induced the additionof 4 acetylated sites to CPS1, while only one of five sites present inthe fed condition disappeared upon fasting (Kim, S. C. et al., 2006).

The evidence for another possible SIRT5 acetylated substrate, cytochromec, is also equivocal (Huang et al., 2010; Gertz and Steegborn, 2010).While SIRT5 has been shown to deacetylate cytochrome c in vitro(Schlicker et al., 2008), there is conflicting data regarding whether itcan localize to the same sub-mitochondrial compartment as cytochrome c,the intermembrane space (Schlicker et al., 2008; Nakamura et al., 2008;Nakagawa et al., 2009). Cytochrome c is a component of the respiratoryelectron transport chain and release of cytochrome c from themitochondrial intermembrane space to the cytoplasm promotes apoptosis(programmed cell death). Overexpression of SIRT5 in cerebellar granuleneurons is pro-apoptotic, consistent at least with a possible SIRT5regulatory role in the latter of these two processes, apoptosis (Pfisteret al., 2008). A regulatory SIRT5 role in respiration has also beensuggested (Gertz and Steegborn, 2010).

An alternative view of SIRT5's physiological function is that it mayprimarily involve catalysis of reactions other than deacetylation.SIRT5's deacetylase activity is detectable but weak with an acetylatedhistone H4 peptide (North et al., 2005) and with chemically acetylatedhistones or bovine serum albumin (Schuetz et al., 2007). The catalyticefficiency of SIRT5 with an acetylated histone H3 peptide(k_(cat)/K_(m)=3.5 s⁻¹ M⁻¹) is orders of magnitude lower than severalhuman and yeast sirtuins (SIRT1, SIRT2, Sir2, Hst2) and more than20-fold lower than the next weakest deacetylase tested, human SIRT3 (Duet al., 2009). Although there is a seeming conflict between the idea ofSIRT5 as a non-deacetylase and its effects on CPS1, it should be notedthat the rate of SIRT5 deacetylation of CPS1 has not been quantified;the deacetylation was only shown in qualitative way by western blottingwith anti-acetyllysine (Nakagawa et al., 2009). Further, although SIRT5performs an NAD⁺-dependent activation of CPS1 and an NAD⁺-dependentdeacetylation of CPS1, no mechanistic link between the deacetylation andthe activation has been established. The in vitro SIRT5/CPS1 activationexperiments were performed with crude mitochondrial matrix lysates fromSIRT5 knockout mice serving as the CPS1 source (Nakagawa et al., 2009).Conceivably, the CPS1 harbored another modification, in addition toacetylation, that SIRT5 reversed in an NAD⁺-dependent reaction.Consistent with this possibility is recently presented evidence thatmitochondrial proteins are lysine-succinylated and that SIRT5 candesuccinylate peptides with efficiencies similar to the deacetylationefficiencies of human SIRTs 1-3 (Lin, 2010).

The activity of lysine deacetylases (class I and II HDACs and sirtuins(class III HDACs)) can be conveniently measured with syntheticsubstrates of the general structure X-Lysine(ε-acetyl)-F, where F is afluorophore or other moiety for which a measurable signal increasesafter cleavage of its direct covalent bond to the carboxyl of lysine andX may be an N-terminal blocking group such as acetyl (Ac) or a peptidesequence (for single-lysine substrates see Hoffman et al., 1999; EnzoLife Sciences Instruction Manual for BML-AK500; Zhou et al., 2001;Bitterman et al., 2002). For longer peptide substrates see U.S. Pat.Nos. 7,033,778; 7,256,013; Howitz et al., 2003. A signal proportional todeacetylation is generated by virtue of the fact that trypsin, amongother lysyl-specific peptidases, will not cleave amide bonds on thecarboxyl side of lysine if the ε-amino of the lysine side-chain ismodified by an acetyl function (Pantazis and Bonner, 1981; Brownlee etal., 1983). A homogenous, endpoint deacetylase assay can thus consist ofa two-step procedure in which the deacetylase is first allowed to act onthe substrate and signal is then generated in a second step in whichtrypsin selectively cleaves the deacetylated substrate molecules. Acontinuously coupled version of this assay procedure has been describedin which the deacetylase, the substrate, and trypsin are all present insame reaction mixture during the deacetylation reaction (Schultz et al.,2004). It should be noted that not all modifications of the lysineε-amino function result in elimination of trypsin cleavability at thelysine carboxyl. Trypsin will cleave at a reduced but significant rateat N^(ε)-monomethyllysine residues (Benoiton and Deneault, 1966; Seelyand Benoiton, 1970; Martinez et al., 1972; Joys and Kim, 1979), whileN^(ε),N^(ε)-dimethyllysine residues are resistant to trypsin cleavage(Poncz and Dearborn, 1983).

Although “X-Lysine(ε-acetyl)-F” substrates are widely used for the assayof various HDAC and sirtuin isoforms, assay of SIRT5 has beenproblematic because the efficiency of SIRT5 deacetylation of suchsubstrates is extremely poor. For example, it has been asserted thatSIRT5 “does not” deacetylate the p53 peptide substrateAc-Arg-His-Lys-Lys(ε-acetyl)-AMC (Nakagawa et al., 2009). While SIRT5will in fact deacetylate this peptide, significant levels ofdeacetylation require either a combination of high peptide substrateconcentration (e.g. 500 high concentration of the cosubstrate NAD⁺ (1 to5 mM) and large quantities of enzyme (˜5 μg/50 μl assay=˜3 μM SIRT5)(U.S. Patent Application Publication 20060014705) or the addition of asirtuin activator such as resveratrol (Id.). Such conditions presentsevere practical problems for SIRT5 assays, particularly in drugdiscovery applications such as the screening of chemical libraries forSIRT5 inhibitor or activator “lead compounds” and the subsequent roundsof inhibitor/activator structure-activity relationship (SAR)characterization and chemical synthetic compound improvement. Forexample, high concentrations of the “X-Lysine(ε-acetyl)-F” typefluorogenic substrates produce a high background fluorescence in allsamples. High fluorescence background increases the difficulty ofobserving statistically significant differences among positive controls,negative controls and inhibitor/activator “hits” (Zhang et al., 1999).Further, the lower limit for determining an enzyme inhibitor's IC₅₀(concentration at which the inhibitor lowers enzyme activity to 50% ofthe uninhibited control sample) is ½ the enzyme concentration (Copeland,2000; Inglese et al., 2008). Thus, the use of a high enzymeconcentrations in an assay impedes the ability to quantitativelydistinguish high and low potency inhibitors/activators and consequentlyinterferes with chemical synthetic efforts to optimize pharmaceuticallead compounds.

The present invention provides compositions and methods which solvethese problems for SIRT5 by, for example, enabling assays to beperformed at drastically lower enzyme concentrations (<20 ng/50<12 nM)and at lower fluorogenic substrate concentrations (<50 μM), whichproduce lower fluorescent background levels. Substrates for detectingother enzymes that remove modifications of ε-amino moieties are alsoprovided.

BRIEF SUMMARY OF THE INVENTION

Provided herein are compounds and methods useful for detecting enzymesthat remove formyl, succinyl, methyl succinyl, or myristoyl moietiesfrom ε-amino lysine moieties of proteins. In some embodiments, acompound is provided that comprises the structure:

wherein SIG is a signaling molecule; m is an integer from 1 to about 10;R¹ is NH, O, S or SO₂; R² is a hydrogen, a halogen, an isothiocyanogroup (SNC), a sulfonate group (SO₃R⁴), a sulfate group (OSO₃R⁴), acarboxyl group (CO₂H), a carbonyl group (COR⁴), an amido group (CONR⁴ ₂or NR³COR⁴), a carbamate group (NR⁴CO₂R⁴), a phosphate group (OPO₃R⁴ ₃),a phosphonate group (PO₃R⁴ ₂), an amino group (NR⁴ ₂), an alkoxy group(OR⁴), a thiol group (SR⁴), a sulfoxy group (SOR⁴), a sulfone group(SO₂R⁴), a sulfonamide group (SO₂NR⁴ ₂), a phosphino group (PR⁴ ₂), asilane group (SiR⁴ ₃), an oligopeptide sequence of 1-20 modified orunmodified amino acids or amino acid substitutes, a protein, aglycoprotein or a lipoprotein; each R⁴ is independently a hydrogen, 1 to3 halogen atoms, a substituted or unsubstituted C₁-C₁₀ straight-chain,branched or cyclic alkyl, alkenyl or alkynyl group wherein one or moreC, CH or CH₂ groups may be substituted with an O atom, N atom, S atom,or NH group, an unsubstituted or substituted aromatic group wherein oneor more C, CH or CH₂ groups may be substituted with an O atom, N atom, Satom, or NH group; and R³ is a formyl, a succinyl, a methyl succinyl, ora myristoyl.

In other embodiments, a kit is provided that comprises the abovecompound, with instructions for determining the presence of the enzyme.

In further embodiments, a method is provided for determining whether asample has an enzyme that removes a moiety from an ε-amino of a lysine,wherein the moiety is a succinyl, a methyl succinyl, a formyl, or amyristoyl. The method comprises (a) combining the sample with the abovecompound to make a sample-compound mixture, wherein R³ of the compoundis the moiety; (b) incubating the sample-compound mixture underconditions and for a time sufficient to allow the enzyme to remove theR³; and (c) determining whether the R³ is removed from the compound. Inthese methods, removal of R³ from the compound indicates that the samplehas the enzyme.

Additionally provided is a method of determining whether a moleculeinhibits an enzyme that removes a moiety from an ε-amino of a lysine,wherein the moiety is a succinyl, a methyl succinyl, a formyl, or amyristoyl. The method comprises (a) combining the enzyme and themolecule with the above compound to make an enzyme-molecule-compoundmixture, wherein R³ of the compound is the moiety; (b) incubating theenzyme-molecule-compound mixture under conditions and for a timesufficient for the enzyme to remove the moiety in the absence of themolecule; and (c) determining whether the R³ is removed from thecompound to an equivalent degree that R³ would be removed from thecompound in the absence of the molecule. In these methods, the failureof the removal of R³ from the compound to an equivalent degree as in theabsence of the molecule indicates that the molecule is an inhibitor ofthe enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a reaction schematic for NAD⁺-Dependent SIRT5 desuccinylationof the substrate N-(α-acetyl-Lysine(ε-succinyl))-AMC (Compound 1) andits detection by specific trypsin release of AMC from the desuccinylatedproduct N-(α-acetyl-Lysine)-AMC (Compound 3).

FIG. 2 is a graph showing that trypsin treatment releases fluorescentAMC from Ac-Lys-AMC but not Ac-Lys(Succinyl)-AMC. Samples (100 μl) of 1μM Ac-Lys-AMC and 1 μM Ac-Lys(Succinyl)-AMC in a buffered solution (50mM Tris/HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂) were treatedwith bovine pancreatic trypsin (2 mg/ml) for the indicated times at roomtemperature. Samples were in the wells of a 96-well, ½-area whitemicroplate and fluorescence was measured at 1 min. intervals at theexcitation and emission wavelengths for free AMC (Excitation: 360 nm;Emission: 460 nm) in a Synergy 2 microplate reading fluorimeter(BioTek), Gain 40. The data points at 0 min. represent the fluorescenceof equivalent 1 μM Ac-Lys(Succ.)-AMC and 1 μM Ac-Lys-AMC samples in theabsence of trypsin.

FIG. 3 is graphs showing that SIRT5 catalyzes NAD⁺-dependentdesuccinylation of Ac-Lys(ε-succinyl)-AMC far more efficiently thanNAD⁺-dependent deacetylation of Ac-Arg-His-Lys-Lys(ε-acetyl)-AMC. Fiftyμl reactions in Assay Buffer (50 mM Tris/HCl, pH 8.0, 137 mM NaCl, 2.7mM KCl, 1 mM MgCl₂, 1 mg/ml BSA) included 50 μM of eitherAc-Lys(ε-succinyl)-AMC or Ac-Arg-His-Lys-Lys(ε-acetyl)-AMC, and, whereindicated, 500 μM NAD⁺ and 5 μg recombinant human SIRT5 (Enzo LifeSciences Cat. # BML-SE555). After 60 min. at 37° C., reactions werestopped and AMC cleaved from deacetylated/desuccinylated substrate byaddition of 50 μl of “Developer” (4 mg/ml trypsin, 2 mM nicotinamide inAssay Buffer). AMC fluorescence was read in a Cytofluor II plate-readingfluorimeter (Perseptive Biosystems) at wavelengths 360 nm(excitation)/460 nm (emission), Gain 54. Data represent the mean of two(No Enzyme) or three (+SIRT5) determinations and error bars the standarddeviations. Panels A and B present the same data, but with the+SIRT5/+NAD⁺/Ac-Lys(ε-succinyl)-AMC bar omitted from B to show thedetail on the remaining bars. Statistically significant differences(Student's t-test) are indicated by asterisks as follows: *: p<0.02 vs.corresponding No Enzyme control; **: p<0.001 vs. corresponding −NAD⁺control; ***: p<3×10⁻⁶ vs. both No Enzyme and −NAD⁺ controls.

FIG. 4 is a graph showing that Ac-Lys(Succ.)-AMC enables SIRT5 assay atlow enzyme concentrations. Fifty μl reactions in Assay Buffer (50 mMTris/HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1 mg/ml BSA)included 2 or 10 μM Ac-Lys(ε-succinyl)-AMC, 500 μM NAD⁺ and, whereindicated, 5 ng recombinant human SIRT5 (Enzo Life Sciences Cat. #BML-SE555). After 20 min. at 37° C., reactions were stopped and AMCcleaved from deacetylated/desuccinylated substrate by addition of 50 μlof “Developer” (4 mg/ml trypsin, 2 mM nicotinamide in Assay Buffer). AMCfluorescence was read in a Synergy 2 plate-reading fluorimeter (BioTek)at wavelengths 360 nm (excitation)/460 nm (emission), Gain 40. Datarepresent the mean of two (No Enzyme) or three (5 ng SIRT5)determinations and error bars the standard deviations. Statisticallysignificant differences (Student's t-test) are indicated by asterisks asfollows: *: p=0.002; **: p=0.009, each with respect to the corresponding“No SIRT5” samples.

FIG. 5 is graphs showing dependence of SIRT5 initial rate kinetics onthe concentrations of Ac-Lys(Succ.)-AMC and Ac-Lys(Ac.)-AMC. Fifty μldesuccinylation or deacetylation reactions were performed as describedearlier (FIG. 4), with the indicated concentrations of Ac-Lys(Succ.)-AMCor Ac-Lys(Ac.)-AMC and 1 mM NAD⁺, for 20 min. at 37° C. Reactions withAc-Lys(Succ.)-AMC contained 10 ng SIRT5 and those with Ac-Lys(Ac.)-AMCcontained 5 SIRT5. Reactions were stopped and AMC cleaved fromdeacetylated or desuccinylated substrate by trypsin treatment asdescribed earlier (FIG. 4). AMC fluorescence was read in a Synergy 2plate-reading fluorimeter (BioTek) at wavelengths 360 nm(excitation)/460 nm (emission), Gain 40. Data represent the mean of thedifferences of three determinations with enzyme (10 ng or 5 SIRT5) fromthe mean of two no enzyme samples for each substrate concentration.Error bars are the standard deviations from those means. Fluorescencedifferences were converted to specific activities (pmol/min/μg) bymeasuring the fluorescence increase due to the addition a standardsolution of AMC (5 μl of a 3 μM solution=150 pmol). Panel A shows thecurve for Ac-Lys.(Succ.)-AMC and kinetic parameters obtained from anon-linear least-squares fit to the Michaelis-Menten equation (MicrosoftXL Solver tool). Panel B shows a the Ac-Lys(Ac.)-AMC data from Panel Awith a 3333-fold expanded y-axis and a linear least-squares fit to thedata.

FIG. 6 is a graph showing the dependence of SIRT5 initial rate kineticsof Ac-Lys(Succ.)-AMC desuccinylation on the concentration of NAD⁺.Reaction conditions were as described for FIG. 5 but with a constantAc-Lys(Succ.)-AMC concentration of 0.5 mM and the indicatedconcentrations of NAD⁺. Data analysis and determination of kineticparameters were as described (FIG. 5).

FIG. 7 is a graph showing the inhibition of SIRT5 desuccinylation ofAc-Lys(Succ.)-AMC by suramin. Desuccinylation reaction conditions wereas described (FIG. 5), but done for 60 min. at 37° C., with theindicated concentrations of suramin (ENZO Life Sciences Cat. #ALX-430-022) and with constant concentrations of 50 μMAc-Lys(Succ.)-AMC, 500 μM NAD⁺ and 12 nM SIRT5 (20 ng/50 μl). Signaldevelopment (trypsin treatment) and fluorescence readings were done asdescribed, as were the conversion of fluorescence increases to rates inunits of pmol/min/μg (FIG. 5). Data points represent the mean of threedeterminations and the error bars are the standard deviations from thosemeans. The dose-response curve was derived from a least-squares fit to athree parameter Hill-Slope model (bottom fixed at 0 pmol/min/μg),y=top/(1+(x/IC₅₀)^(slope)). The fitted parameters were top=127pmol/min/μg, IC₅₀=27.3 μM and slope=2.37 (‘Solver’ tool, Microsoft XL).

FIG. 8 is a graph showing inhibition of SIRT5 desuccinylation ofAc-Lys(Succ.)-AMC by nicotinamide. All reaction conditions andprocedures were as described for the suramin inhibition study (FIG. 7)but reactions were done instead with the indicated concentrations ofnicotinamide (ENZO Life Sciences Cat. # BML-KI283). Data pointsrepresent the mean of three determinations and the error bars are thestandard deviations from those means. The dose-response curve wasderived from a least-squares fit to a three parameter Hill-Slope model(bottom fixed at 0 pmol/min/μg), y=top/(1+(x/IC₅₀)^(slope)). The fittedparameters were top=90.2 pmol/min/μg, IC₅₀=29.0 μM and slope=0.98(‘Solver’ tool, Microsoft XL).

FIG. 9 is graphs showing the high specific activity of SIRT5 catalyzedNAD⁺-dependent desuccinylation of Ac-Lys(ε-succinyl)-AMC compared withminor activities of non-SIRT HDACs and HeLa nuclear extract. Initialrate activities of the indicated enzyme were determined with 50 μMAc-Lys(Succ.)-AMC for HDACs 1-11 and HeLa nuclear extract. SIRT5activity was determined with 50 μM Ac-Lys(Succ.)-AMC plus 500 μM NAD⁺.Panels A and B present the same data, but with the SIRT5 bar omittedfrom B in order to display the remaining bars at a 240-fold higherscale.

FIG. 10 is graphs showing that intact HeLa cells do not significantlydesuccinylate Ac-Lys(ε-succinyl)-AMC under the conditions that allowdeacetylation of Ac-Lys(ε-acetyl)-AMC. HeLa cells were cultured to 95%confluence in the wells of ½-volume 96-well plates. Either 200 μMAc-Lys(Succ.)-AMC (Panel A) or 200 μM Ac-Lys(Ac.)-AMC (Panel B) wasadded to the medium, either alone or with trichostatin A (TSA, 1 μM) ornicotinamide (NAM, 1 mM). After the indicated time, cells were lysedwith detergent and trypsin added to release AMC from desuccinylated ordeacetylated substrate and then fluorescence read (Ex.360 nm/Em. 460nm). For “0 hr.” samples, substrate, inhibitors, lysis buffer andtrypsin were added simultaneously. Bars represent the mean of threedeterminations and the error bars the Standard Deviation from that mean.Statistically significant differences between the fluorescences of “4hr.” and “0 hr.” samples (Student's t-test) are indicated by asterisksas follows: *,p<0.02; **, p<0.002.

FIG. 11 is graphs showing that HeLa cell extracts haveAc-Lys(ε-succinyl)-AMC desuccinylation activity that is partiallyNAD⁺-dependent/suramin-sensitive (SIRT5) and partially TSA-sensitive(class I HDACs). HeLa cell extracts were prepared by hypotonic/detergentlysis (0.5% NP-40) and assayed for Ac-Lys(ε-succinyl)-AMCdesuccinylation activity (extract equivalent to 28×10⁴ cells per assaywell). Panel A shows cell extracts incubated with 50 μMAc-Lys(Succ.))-AMC and incubated for 0 hr. or 2 hr. (37° C.) withindicated additions. “No Lysate” (NL) sample contained only the bufferof equivalent to that of the cell extracts and these samples wereincubate 2 hr. T=TSA (1 μM) and S=suramin (200 μM). NAD⁺, when present,was 500 μM. At the end of the substrate incubations, AMC was cleavedfrom desuccinylated substrate by trypsin treatment and fluorescencedetermined (Ex. 360 nm, Em. 460 nm). Bars represent the mean of twodeterminations and the error bars the standard deviations from thosemeans. Panel B shows the fluorescence increases from 2 hr. of extractactivity were calculated by subtracting the corresponding 0 hr. datafrom the 2 hr. data in Panel A. For the No NAD⁺/Suramin data there wereno 0 hr. samples and the corresponding NL value was subtracted instead.Bars are labeled to indicate which inhibitor(s), if any, were present.

FIG. 12 is graphs showing that SIRT5 catalyzes NAD⁺-dependentdesuccinylation of (Ac-Lys(Succ.))₂-R110 far more efficiently thanNAD⁺-dependent deacetylation of (Ac-Lys(Ac.))₂-R110. Fifty μl reactionsin Assay Buffer (50 mM Tris/HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mMMgCl₂, 1 mg/ml BSA) included 50 μM of either (Ac-Lys(Succ.))₂-R110 or(Ac-Lys(Ac.))₂-R110, and, where indicated, 500 μM NAD⁺ and 5 μgrecombinant human SIRT5 (Enzo Life Sciences Cat. # BML-SE555). After 60min. at 37° C., reactions were stopped and the Ac-Lys cleaved from theR110 (rhodamine green) substrate by addition of 50 μl of “Developer” (4mg/ml trypsin, 2 mM nicotinamide in Assay Buffer). Fluorescence was readin a Cytofluor II plate-reading fluorimeter (Perseptive Biosystems) atwavelengths 485 nm (excitation)/530 nm (emission), Gain 37. Datarepresent the mean of two (No Enzyme) or three (+SIRT5) determinationsand error bars the standard deviations. Panels A and B present the samedata, but with the +SIRT5/+NAD⁺/(Ac-Lys(Succ.))₂-R110 bar omitted from Bto show the detail on the remaining bars. Statistically significantdifferences (Student's t-test) are indicated by asterisks as follows:**: p<3×10⁻⁵ vs. corresponding +NAD⁺/No Enzyme control; ***: p<2×10⁻⁶vs. corresponding +NAD⁺/No Enzyme control.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Additionally, the use of “or” is intended to include“and/or”, unless the context clearly indicates otherwise.

The present invention is directed to compounds and methods useful fordetecting enzymes that remove formyl, succinyl, methyl succinyl, ormyristoyl moieties from ε-amino lysine moieties of proteins. Methods foridentifying inhibitors of those enzymes are also provided.

Thus, in some embodiments, a compound is provided that comprises thestructure:

wherein

-   -   SIG is a signaling molecule;    -   m is an integer from 1 to about 10;    -   R¹ is NH, O, S or SO₂;    -   R² is a hydrogen, a halogen, an isothiocyano group (SNC), a        sulfonate group (SO₃R⁴), a sulfate group (OSO₃R⁴), a carboxyl        group (CO₂H), a carbonyl group (COR⁴), an amido group (CONR⁴ ₂        or NR³COR⁴), a carbamate group (NR⁴CO₂R⁴), a phosphate group        (OPO₃R⁴ ₃), a phosphonate group (PO₃R⁴ ₂), an amino group (NR⁴        ₂), an alkoxy group (OR⁴), a thiol group (SR⁴), a sulfoxy group        (SOR⁴), a sulfone group (SO₂R⁴), a sulfonamide group (SO₂NR⁴ ₂),        a phosphino group (PR⁴ ₂), a silane group (SiR⁴ ₃), an        oligopeptide sequence of 1-20 modified or unmodified amino acids        or amino acid substitutes, a protein, a glycoprotein or a        lipoprotein;    -   each R⁴ is independently a hydrogen, 1 to 3 halogen atoms, a        substituted or unsubstituted C₁-C₁₀ straight-chain, branched or        cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH        or CH₂ groups may be substituted with an O atom, N atom, S atom,        or NH group, an unsubstituted or substituted aromatic group        wherein one or more C, CH or CH₂ groups may be substituted with        an O atom, N atom, S atom, or NH group; and    -   R³ is a formyl, a succinyl, a methyl succinyl, or a myristoyl.

When the above compound is combined with an enzyme that removes the R³moiety under the proper conditions (e.g., in the presence of NAD⁺ whenthe enzyme is a class III HDAC [sirtuin]), the enzyme will remove the R³moiety, leaving an unaltered lysine residue. The resulting compound canbe identified by any method known in the art, for example by massspectroscopy, an immunoassay with an antibody that can distinguishbetween the compound with the R³ group and without it, or an appropriatechromatographic method. In some embodiments, the compound without the R³group is a substrate for a peptidase while the compound with the R³group is not a peptidase substrate, such that removal of the R³ groupfollowed by treatment with the peptidase leaves SIG-R¹. In variousembodiments, such as where SIG is a fluorescent or a luminescent moiety,SIG-R¹ has an increased signal (e.g., increased fluorescence orluminescence) than the intact compound. Under those conditions, theremoval of the R³ group is detected by addition of the peptidase,releasing SIG-R¹ and providing an increased signal. This increasedsignal thus establishes the presence of the enzyme that removes the R³group.

Thus, in some embodiments, the compound is a substrate for an enzymesuch that the enzyme cleaves R³ from the compound allowing a peptidaseto cleave the resulting molecule between the R¹ and the CO moieties,such that SIG generates an increased signal relative to the signalgenerated with the compound. In these embodiments, the peptidase cannotcleave the compound comprising R³. See Example.

The peptidase for these embodiments can be any peptidase that is capableof cleaving the compound without the R³ group to release SIG-R¹ but notcapable of cleaving the compound with the R³ group. A nonlimitingexample of such a peptidase, where the R³ group is a succinyl, a methylsuccinyl, or a myristoyl group, is trypsin.

Where the R³ group is a formyl group, trypsin cannot be used asdescribed for the other R³ groups, since trypsin is capable of cleavingthe compound between the R¹ and CO moieties even when the formyl groupis not removed, albeit at a slower rate. See Benoiton and Deneault(1966), Seely and Benoiton (1970); Martinez et al. (1972); and Joys andKim (1979). Under those circumstances, the use of trypsin would have tobe modified such that the trypsin concentration is low enough such thatthe cleavage kinetics could be observed, where faster cleavage wouldindicate the elimination of the formyl R³ group and slower cleavagewould indicate the retention of the formyl R³ group. Alternatively,another peptidase could be used when the R³ group is a formyl group,e.g., a peptidase that will not cleave the compound between the R¹ andCO moieties when the formyl is present but will when the formyl iscleaved. A likely example of such an enzyme is endoproteinase Lys-C,which is unable to cleave monomethyllysine residues in proteins.

In some embodiments of these compounds, R¹ is NH. In other embodiments,m is 1 or 2.

In various embodiments, the R² moiety is a chemical protecting group.Such a protecting group is useful for the synthesis of the compound,since blocking the α-amino group allows the unambiguous addition of theR³ moiety to the ε-amino group, without concern that the R³ moiety wouldbe inadvertently added to the α-amino group. Any protecting group knownin the art as useful for protecting amino moieties could be useful here.Nonlimiting examples include FMOC, acetyl, benzoyl, Aloc, arysulfenyl,benzyl, BOM, BOC, carbobenzyloxy, diphenylmethylene, DMPM, EE, PMB,methoxycarbonyl, MeOZ, MoM, PMP, Noc, Nosyl, Nps, PhFI, Psec, pixyl,tosyl, Tsoc, Troc, trifluoroacetyl, TIPS, TMS, SES, Teoc, SEM, andTrity. In some embodiments (as in the Example) R² is an acetyl group.

Examples of the compounds include

In various embodiments, m is 1 or 2.

These compounds are useful for detecting any enzyme that removes the R³group. In some embodiments, the enzyme is a histone deacetylase (HDAC).As shown in Table 2 below, HDAC2 and HDAC3/NCOR1 complex have someactivity removing the R³ moiety when that moiety is a succinyl group.Additionally, as shown in Table 3 below, HDAC1, HDAC3, and particularlyHDAC2 and HDAC3/NCOR1 complex have activity removing the R³ moiety whenthat moiety is a myristoyl or a methyl succinyl group. Additionally,HDAC9 has activity removing a methyl succinyl R³ group.

In some of these embodiments, the HDAC is a sirtuin (a class III HDAC).As shown in Table 3 below, SIRT1, SIRT3 and SIRT6 have activity removinga myristoyl R³ group. Additionally, as discussed extensively in theExample and shown in Table 2, SIRT5 has activity removing a succinyl R³group that is about two orders of magnitude greater than its deacetylaseactivity.

The signal, SIG, can be any chemical compound that has decreasedfluorescence, luminescence or color intensity when functionalized withone or more of the

groups. Ideally, SIG is non-fluorescent, non-luminescent and colorlesswhen the group is attached and intensely fluorescent, luminescent orcolored when the group is removed. Additionally, SIG should contain orshould be readily modified to contain reactive functionalities, asfurther discussed below, to which the above group could be attached toform a probe.

The invention is not narrowly limited to the use of any particular SIG.In various embodiments, SIG is a chromophore, a fluorophore, aluminescent moiety, an enzyme, a catalytic antibody, a ribozyme or apro-enzyme.

In some embodiments, SIG is a fluorophore. Any fluorophore now known orlater discovered can be utilized in these compounds. Examples of usefulfluorophores include without limitation a symmetric or asymmetriccyanine dye, a merocyanine dye, a styryl dye, an oxazine dye, a xanthenedye, a coumarin dye or an iminocoumarin dye.

One class of the signal molecule, SIG, useful in the invention has axanthene backbone shown in Scheme I below. The structures include bothclassical xanthene dyes and their lactone forms (Structures A and B,respectively) as well as aphenylic counterparts, which have theirappended phenyl ring missing (Structures C).

The substituent R⁵ in Scheme I represents a variety of functionalitieswhere at least one R⁵ is a reactive group, which allows the attachmentof the

group and, if desired, at least one other R⁵ is a reactive group, whichallows the attachment of a protecting group to prevent attachment ofadditional groups, if preferred. The R⁵s may be structurally the same ordifferent and there may be several of them per ring. Also, some of therings may not have any R⁵s attached. Suitable examples of R⁵ include,but are not limited to hydrogen, a halogen (F, Cl, Br, I), a nitro group(NO₂), a nitroso group (NO), a hydroxylamino group (NHOH), a cyano group(CN), an isocyano group (NC), a thiocyano group (SCN), an isothiocyanogroup (SNC), an azido group (N₃), a trihalomethyl group (CX₃, where X isa halogen), a sulfonate group (SO₃R⁶), a sulfate group (OSO₃R⁶), acarboxyl group (CO₂H), a carbonyl group (COW), an ester group (CO₂R⁶ orOCOR⁶), an amide group (CONR⁶ ₂ or NR⁶COR⁶), a carbamate group (NR⁶CO₂R⁶or OCONR⁶ ₂), a phosphate group (OPO₃R⁶ ₃), a phosphonate group (PO₃R⁶₂), an amino group (NR⁶ ₂), an alkoxy group (OR⁶), a thiol group (SR⁶),a sulfoxy group (SOR⁶), a sulfone group (SO₂R⁶), a sulfonamide group(SO₂NR⁶ ₂), a phosphino group (PR⁶ ₂), a silane group (SiR⁶ ₃), anoptionally substituted straight-chain, branched or cyclic alkyl, alkenylor alkynyl group wherein one or more C, CH or CH₂ groups can be replacedwith O atom, N atom, S atom, NH group, CO group, OCO group, CONR⁶ group,or an optionally substituted aromatic group. In these embodiments, eachR⁶ is independently hydrogen, an optionally substituted straight-chain,branched or cyclic alkyl, alkenyl or alkynyl group wherein one or moreC, CH or CH₂ groups can be replaced with O atom, N atom, S atom, NHgroup, CO group, OCO group, CONR⁶ group, or an optionally substitutedaromatic group.

Two or more R⁵ groups in these fluorophores can be linked together toform rings containing one or more of the same or different heteroatoms,such as O, N or S.

Substituents R⁵ in these fluorophores that are not directly involved inattachment of self-immolative or urea-containing groups may be presentin the molecule for other reasons. These reasons may includemodification of spectroscopic characteristics of the dye, itssolubility, chemical stability, charge, or photobleaching resistance.Some R⁵ substituents may be useful for binding to a biomolecule orstructure to be studied, such as nucleic acid, protein or lipid.

As discussed above, one of the R⁵ or R⁶ groups is, or can be substitutedto contain, a reactive group thereby allowing the dyes of the presentinvention to be attached to an

group. Examples of reactive groups that may find use in the presentinvention can include but not be limited to a nucleophilic reactivegroup, an electrophilic reactive group, a terminal alkene, a terminalalkyne, a platinum coordinate group or an alkylating agent.

There are a number of different electrophilic reactive groups that mayfind use in these embodiments. Examples include but not be limited toisocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine,4,6-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine,mono- or di-halogen substituted diazine, maleimide, haloacetamide,aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester,hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol,azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal and aldehyde groups.Nucleophilic reactive groups can include but not be limited to reactivethiol, amine and hydroxyl groups. For purposes of synthesis of dyes,reactive thiol, amine or hydroxyl groups can be protected during varioussynthetic steps and the reactive groups generated after removal of theprotective group.

One class of xanthene fluorophores useful in the present inventionincludes but not limited to rhodamine and rhodamine derivatives, such asPennsylvania Green, Tokyo Green, Oregon Green, Singapore Green, androsamines and rhodols and their derivatives. Some of these derivativesare shown below in Scheme II. The rhodamine, rosamine and rhodolbackbone structures can be extended by adding additional rings as shownin Scheme III, or their appended phenyl ring might be missing to formaphenylic counterparts.

Another class of fluorescent dyes pertinent to the present invention isderivatized from the aforementioned rhodamines, rosamines and rhodolsand can be represented by the general structures shown in Scheme IV.

The substituent R⁵ in Scheme IV is defined as described for Scheme I.The moiety A can be oxygen or sulfur while Z can be oxygen, sulfur ornitrogen unsubstituted or substituted with a group Y. The group Y, inturn, can be hydrogen, an optionally substituted straight-chain,branched or cyclic alkyl, alkenyl or alkynyl group wherein one or moreC, CH or CH₂ groups can be replaced with O atom, N atom, S atom, NHgroup, CO group, OCO group, CONR³ group, or an optionally substitutedaromatic group. Y can also be another nitrogen, oxygen or sulfur atomsubstituted with hydrogen or an optionally substituted straight-chain,branched or cyclic alkyl, alkenyl or alkynyl group wherein one or moreC, CH or CH₂ groups can be replaced with O atom, N atom, S atom, NHgroup, CO group, OCO group, CONR³ group, or an optionally substitutedaromatic group. The substituent Y can be a part of an aliphatic oraromatic cyclic structure such as morpholine, piperidine, pyrrolidine,piperazine, imidazole, triazole, oxazole, thiazole and others known inthe art. Additionally, both Z and Y can contain electrophilic ornucleophilic reactive groups defined above.

Yet another class of fluorescent dyes pertinent to the present inventionis based on coumarin and iminocoumarin backbone structure shown inScheme V.

The substituent R⁵ in the Scheme V represents functionalities defined inScheme I above while A can be oxygen atom, O, or imino group, NH. Someof the compounds in this category are shown below in Scheme VI. Thebackbone structure can be extended by adding additional rings, aliphaticor aromatic, substituted or unsubstituted.

In other embodiments of the compounds of the present invention, SIG is aluminescent moiety. Any luminescent moiety, including anychemiluminescent or bioluminescent moieties, now known or laterdiscovered, can be utilized in these embodiments. In some aspects ofthese embodiments, the compound comprises the structure:

wherein

each R⁵ is independently hydrogen, a halogen (F, Cl, Br, I), a nitrogroup (NO₂), a nitroso group (NO), a hydroxylamino group (NHOH), a cyanogroup (CN), an isocyano group (NC), a thiocyano group (SCN), anisothiocyano group (SNC), an azido group (N₃), a trihalomethyl group(CX₃, where X is a halogen); a sulfonate group (SO₃R⁶), a sulfate group(OSO₃R⁶), a carboxyl group (CO₂H), a carbonyl group (COR⁶), an estergroup (CO₂R⁶ or OCOR⁶), an amide group (CONR⁶ ₂ or NR⁶COR⁶), a carbamategroup (NR⁶CO₂R⁶ or OCONR⁶ ₂), a phosphate group (OPO₃R⁶ ₃), aphosphonate group (PO₃R⁶ ₂), an amino group (NR⁶ ₂), an alkoxy group(OR⁶), a thiol group (SR⁶), a sulfoxy group (SOR^(E)), a sulfone group(SO₂R⁶), a sulfonamide group (SO₂NR⁶ ₂), a phosphino group (PR⁶ ₂), asilane group (SiR⁶ ₃), an optionally substituted straight-chain,branched or cyclic alkyl, alkenyl or alkynyl group wherein one or moreC, CH or CH₂ groups can be replaced with O atom, N atom, S atom, NHgroup, CO group, OCO group, CONR⁶ group, or an optionally substitutedaromatic group; and

each R⁶ is independently hydrogen, an optionally substitutedstraight-chain, branched or cyclic alkyl, alkenyl or alkynyl groupwherein one or more C, CH or CH₂ groups can be replaced with O atom, Natom, S atom, NH group, CO group, OCO group, CONR⁶ group, or anoptionally substituted aromatic group.

In some embodiments, the SIG is a fluorescent compound that targets aspecific subcellular organelle, for example the lysosome, mitochondria,vacuole, nucleus or nucleolus. See, e.g., PCT/US10/002494 andPCT/US10/02572 and references cited therein.

Specific examples of the invention compounds, as further described inthe example below include

The first two compounds above are SIRT5 substrates, as they have asuccinyl group as the R³ moiety. See Example, where those two compoundsare Compound 1 (Ac-Lys(Succ.)-AMC) and Compound 2((Ac-Lys(Succ.))₂-R110), respectively. Those compounds can begeneralized as the formulas X-Lysine(ε-succinyl)-F and(X-Lysine(ε-succinyl))₂-F respectively, where, for both Compound 1 andCompound 2, X is an Nα acetyl function (Ac), and F is AMC, for Compound1 and R110 (rhodamine green) for Compound 2. X could, however, bereplaced by other N-terminal protecting groups, such ast-butyloxycarbonyl (Boc) or benzyloxycarbonyl (Cbz) or by peptidesequences of various lengths, while maintaining the essence of the assayprinciple (i.e. desuccinylation-dependent increase in a signal producedby a trypsin treatment, see FIG. 1). For example, the peptide substrateAc-Arg-His-Lys-Lys(ε-acetyl)-AMC (Enzo Life Science Cat. # BML-KI177) isfar more effective as a deacetylation substrate for SIRT5 than isAc-Lys(Ac.)-AMC (Table 2; U.S. Patent Application Publication20060014705). Thus, replacing X in either Compound 1 or Compound 2 withthe sequence Ac-Arg-His-Lys could improve on these single-lysine SIRT5desuccinylation substrates as the equivalent replacement improved on thesingle-lysine deacetylation substrate Ac-Lys(Ac.)-AMC. The sequenceArg-His-Lys-Lys is derived from residues 379-382 of p53 and a fractionof cellular p53 is localized to the mitochondria, including some inassociation with mitochondrial DNA in the matrix (Mahyar-Roemer et al.,2004; de Souza-Pinto et al., 2004; Chen et al., 2006; Bakhanashvili etal., 2008), the submitochondrial compartment containing most, if notall, SIRT5 (Michishita et al., 2005; Nakagawa et al., 2009). Thus it ispossible that the affinity of SIRT5 for the p53 379-382 sequencereflects some in vivo association between the two proteins.

Other candidate amino acid sequences that might function well as the Xgroup in SIRT5 substrates could be sought by means of ananti-succinyllysine antibody. The immunizing antigen for such anantibody may be prepared, for example, by succinylation of the proteinkeyhole limpet hemocyanin (KLH) with succinic anhydride, a procedureequivalent to that used to prepare the immunogens for rabbit polyclonalanti-propionyllysine and anti-butyryllysine antibodies (Enzo LifeSciences Cat. # s BML-SA683, BML-SA682). Production of the sameimmunogen via carbodiimide coupling and its use in preparation of amouse monoclonal anti-succinyllysine have also been described (Kawai etal, 2006). An anti-succinyllysine antibody could be used to prepare anaffinity chromatography matrix to enrich succinylated proteins from amitochondrial or other cellular fraction. The succinylated proteins andthe sequences surrounding their sites of lysine-succinylation could thenbe identified by established chromatographic and mass spectrometricprocedures (Cheng et al., 2009). An anti-succinyllysine antibody couldalso form the basis of desuccinylation assays differing from thefluorometric assays already described (e.g. FIG. 1). SIRT5 or anotherdesuccinylating enzyme would be brought into contact withlysine-succinylated peptides or proteins in the presence of appropriatecofactors (NAD⁺ for SIRT5). The loss of lysine-succinylation would thenbe detected by standard immunochemical means (e.g. western blotting,ELISA).

Although one may yet be identified, there is currently no knownN^(ε)-lysyl succinyltransferase that can perform protein lysylsuccinylation using succinyl-CoA as the succinyl donor, a reactionanalogous to those of the protein (histone) acetyltransferases (HATs);see Hodawadekar and Marmorstein, 2007. However, non-enzymaticsuccinylation of lysine by the peroxidation products of docosahexaenoicacid (DHA) has been demonstrated in vitro with peptides and proteins andthe formation of protein succinyllysine residues in vivo has been shownto occur in DHA-fed mice subjected to oxidative stress (Kawai et al.,2006). Thus, another route to identification of the amino acid sequencesproximal to native lysine succinylation sites would be to exposemitochondrial protein fractions to DHA oxidation products (Id.) and tothen identify the sites by established chromatographic and massspectrometric procedures (Cheng et al, 2009). Since these sites would beexpected to include those that are targets of SIRT5 action in vivo, someof the sequences so identified would likely enhance the SIRT5 activitywhen incorporated into the “X” and/or “F” portions of syntheticsubstrate structures. Note that incorporation of a sequence from theN-terminal side of native SIRT1 deacetylation targets p53 Lys(Ac)-382and histone H4 Lys(Ac)-16 has been shown to enhance activity relative tothe single-lysine substrate Ac-Lys(Ac)-AMC and relative to substratesincorporating sequence from non-targeted sites (Howitz et al, 2003);Appendix E, Enzo Life Sciences Product instruction manual/assay kitprotocol for SIRT1, Cat. # BML-AK555).

For Compound 1 and Compound 2, fitting the general formulasX-Lysine(ε-succinyl)-F and (X-Lysine(ε-succinyl))₂-F, signal generationis due to the bathochromic shift in fluorescence emission upon adesuccinylation-dependent trypsin-catalyzed release of the “F” function,where F is AMC, for Compound 1 and is either R110 (rhodamine green) orthe mono-succinyllysine derivative (X-Lys(Succ.))-F for Compound 2.However, F could be any moiety that undergoes some spectroscopic changeupon the desuccinylation-dependent hydrolysis of its bond with thecarboxyl function of the lysine. For example, F could be p-nitroaniline(pNA), whose absorbance at 405 nm increases after trypsin hydrolysis ofan amide bond between pNA and the lysine carboxyl (Appendix G, Enzo LifeSciences Instruction Manual for BML-AK501). Substrates with such dyes inthe F position could be used in conjunction with one of the previouslylisted charge-neutralizing modifications to the distal carboxyl of thesuccinyl moiety, such as AM-esterification, thus combining an elementthat improves membrane permeability with one enabling targeting toSIRT5's subcellular location in the mitochondria or the nuclear locationof class I HDACs.

For Compounds 1 and 2, and for those described immediately above, a dyegroup F in the structures X-Lysine(ε-succinyl)-F and(X-Lysine(ε-succinyl))₂-F directly forms a direct bond with the carbonylof lysine and provides the assay signal upon desuccinylation-dependenttrypsin cleavage of that bond. A substrate incorporating an equivalentdesuccinylation-dependent signaling system could, alternatively,comprise the following: 1) a spectroscopically detectable function zthat is part of either F or X, but does not form a direct covalent bondto the lysine carbonyl; and 2) a function q that is part of X if z ispart of F or is part of F if z is part of X and which acts to suppressthe detectable signal from z unless a desuccinylation-dependent cleavageof the bond between the lysine carbonyl and F occurs. An example of sucha substrate is the peptide: (5-FAM)-QSTSSHS-K(Succ.)-LMFK(5(6)-TAMRA),(one-letter amino acid code, 5-FAM=5-carboxyfluorescein modifying theN-terminus, 5(6)-TAMRA=5-(and 6)-carboxytetramethylrhodamine modifyingthe ε-amino of the lysine side chain). This peptide comprises theresidues 375-386 of p53, but is modified by R(379)S and K(381)Ssubstitutions to eliminate trypsin-cleavable sites (underlined) and issuccinylated on the ε-amino of K(382). In terms of the generalizedstructure, X-Lysine(ε-succinyl)-F, (5-FAM)-QSTSSHS is X, LMFK(5(6)-TAMRA) is F, z is 5-FAM and q is 5(6)-TAMRA. In the intactpeptide, the fluorescence of the 5-FAM (Ex. 492 nm; Em. 518 nm) isquenched by resonance energy transfer due to proximity and spectraloverlap with 5(6)-TAMRA (Ex. 542 nm; Em. 568). Desuccinylation-dependenttrypsin cleavage after K(382) would produce a fluorescein fluorescencesignal by separating the 5-FAM from the quenching 5(6)-TAMRA.Replacement of trypsin with the similar, but lysine-specific, enzymelysyl endopeptidase (EndoLysC; EC 3.4.21.50) would allow the inclusionof arginine residues (trypsin cleavable sites) in sequence interveningbetween the signaling “z” function and the signal-suppressing “q”function. In the preceding example, the q group suppresses the signalfrom the z group by fluorescence resonance energy transfer, but withdifferent types of q moieties other mechanisms may be employed. Forexample, q could be an affinity tag such as biotin, allowing thephysical removal of the background fluorescence inherent in theremaining succinylated/uncleaved peptide by means of the biotin-bindingprotein streptavidin linked to a solid support such as agarose beads.

The succinylated substrate Ac-Lys(Succ.)-AMC (Compound 1), in additionto providing a highly effective and convenient means of SIRT5 assay,also provided the means to discover a surprisingly strong, TSA-sensitivedesuccinylase activity in HeLa cell extracts, in all likelihood due toclass I HDACs (see FIG. 11, Table 2 and FIG. 9). This substrate couldalso be used in the processes of: 1) purifying and identifying theparticular TSA-sensitive enzyme or enzymes that contribute to thisdesuccinylase activity, 2) locating the subcellular compartment in whichthe activity resides, 3) isolating any multiprotein complexes harboringdesuccinylase activity and 4) determining which protein binding partnersenhance the catalytic activity of the TSA-sensitive desuccinylase(s). Aswill be discussed below, there are reasons to suspect that the ε-aminofunction of the lysine side-chain of proteins in vivo may be subject toa wide variety of novel, non-acetyl acylations in addition tosuccinylation. By replacing the succinyl moiety in Compound 1 orCompound 2 or in any of the structural variants ofX-Lysine(ε-succinyl)-F or (X-Lysine(ε-succinyl))₂-F described above, atool for discovery of deacylase enzymes and the mechanisms that regulatetheir activities (processes 1-4, above) could be constructed for eachtype of NE-acyllysine modification. Once a new deacylase was identified,the corresponding lysine-acylated substrate or substrates could be usedfor the same types of compound screening for drug discovery, kineticcharacterization, mechanistic studies and further assay development asdescribed for the succinyl substrate 1 (FIGS. 4-8, 10, 11).

One reason for the prediction that additionalnon-deacetylase/non-desuccinylase lysyl deacylase activities remain tobe discovered is the documented existence of in vivo modifications tothe ε-amino group of lysine that derive from reaction with oxidativebreakdown products of various biomolecules. For example,3′-formylphosphate, which arises from the 5′-oxidation of deoxyribose inDNA leads to the N^(ε)-formylation of lysines in histones and thisprocess is stimulated by oxidative stress (Jiang et al., 2007).Formylation of the linker histone H1 is especially prevalent (Wisniewskiet al., 2007), but also extends to the N-terminal tails and globulardomains of core histones and other nuclear proteins (Wisniewski et al.,2008). Such modifications would be expected to interfere with chromatinstructure (DNA binding by histone H1 and core histones) and with thesystem of epigenetic lysine modifications (acetylation, methylationespecially in core histone N-terminal tails) that function in theregulation of gene expression. Therefore a lysine deformylase thatfunctions as a repair enzyme in response to oxidative stress damage mustbe considered a distinct possibility and substrates of typeX-Lysine(ε-formyl)-F or (X-Lysine(ε-formyl))₂-F would be useful for thesame processes described above for succinylated substrates.

Peroxides derived from the oxidation of polyunsaturated fatty acids(PUFAs) can react with cellular proteins to form novel NE-acylatedlysine residues (for review see Kato and Osawa, 2010). As noted above,protein lysine residues can be succinylated by reaction with theperoxidation products of DHA, a PUFA (Kawai et al., 2006). In addition,PUFA-derived NE acyl-lysine adducts can include the hexanoyl, glutaroyland azelayl acylations. Similar to the formyl modification, these arethe result of oxidative stress and could be disruptive to proteinfunction. Therefore, lysine deacylases specific for these modificationsand that function as repair enzymes in response to oxidative stressdamage must be considered a distinct possibility. Thus substrates oftype X-Lysine(ε-acyl)-F or (X-Lysine(ε-acyl))₂-F would be useful for thesame discovery processes described above for succinylated substrates.The ε-acyl functions would include the hexanoyl, glutaroyl and azelaylmoieties.

As noted earlier, N^(ε)-acetylation of protein lysine residues iscatalyzed by transferases that use acetyl-CoA as the donor of the acetylgroup. There are twenty-six acyl-coenzyme A synthetases in the humangenome (Watkins et al., 2007) and the Human Metabolome Database lists134 acyl-CoAs that could be the source of novel lysine acylationscatalyzed by hitherto unidentified transferase enzymes (see the websitehmdb.ca/search/search?query=%22CoA %22), exclusive of: 1) CoA itself, 2)acetyl-CoA, propanoyl-CoA, butyryl-CoA (known substrates of theacetyltransferases (HATs)), 3) propinol, propinol adenylate,3-hydroxyvaleric acid (non-CoA metabolites). Each of these may beconsidered a potential acyl donor for transfer to the N^(ε)-aminofunction of protein lysines and consequently substrates of typeX-Lysine(ε-acyl)-F or (X-Lysine(ε-acyl))₂-F, where the ε-acyl functionis any of the 134 cited above, would be useful for the same types ofdiscovery processes described above for succinylated substrates. For oneof these acyl-CoA's, myristoyl-CoA, there is evidence for its use intransfer to specific lysine residues in the precursor protein of tumornecrosis factor α (Stevenson et al., 1992) and in the precursor proteinof interleukin 1α (IL-1α) (Stevenson et al., 1993).

Any of the above-described compounds can be packaged in a kit forcommercial sale. In some embodiments, the kit further comprisesinstructions for determining the presence of the enzyme, or instructionsfor determining the presence of an inhibitor of the enzyme. In some ofthese embodiments, the enzyme is a histone deacetylase (HDAC), forexample, a sirtuin, e.g., SIRT5. An example of a compound that couldusefully be packed in a kit is

More specific examples include

These kits can also contain other reagents that are useful fordetermining the presence of the enzyme or an inhibitor of the enzyme.Non-limiting examples of such reagents include a standard of a knownconcentration of the enzyme and/or an inhibitor of the enzyme, apeptidase (e.g., trypsin), luciferase if SIG is a luciferase substrate,and/or appropriate buffers.

The present invention is also directed to a method of determiningwhether a sample has an enzyme that removes a moiety from an ε-amino ofa lysine, where the moiety is a succinyl, a methyl succinyl, a formyl,or a myristoyl. The method comprises

(a) combining the sample with any of the above-described compounds tomake a sample-compound mixture, where R³ of the compound is the moiety;

(b) incubating the sample-compound mixture under conditions and for atime sufficient to allow the enzyme to remove the R³; and

(c) determining whether the R³ is removed from the compound. In thesemethods, removal of R³ from the compound indicates that the sample hasthe enzyme.

The proper conditions for incubating the sample-compound mixture couldbe determined for any particular enzyme without undue experimentation.For example, when the enzyme is a class III HDAC [sirtuin]), NAD⁺ shouldbe present.

The determination of whether the R³ moiety is removed can be by anymeans known in the art, for example by mass spectroscopy, an immunoassaywith an antibody that can distinguish between the compound with the R³group and without it, or an appropriate chromatographic method. In someembodiments, the compound is a substrate for a peptidase after theenzyme cleaves R³ from the compound but not if the R³ is not removedfrom the compound. In such as case, the determining step furthercomprises

(i) adding the peptidase to the mixture for a time sufficient for thepeptidase to cleave the resulting molecule between the R¹ and the COmoieties, such that SIG generates an increased signal relative to thesignal generated with the compound; and

(ii) determining whether SIG generates an increased signal relative tothe signal generated with the compound. Here, an increased signal (e.g.,greater fluorescence) indicates that the sample has the enzyme.

The present methods are not narrowly limited to the use of anyparticular peptidase. In various embodiments, the peptidase is a trypsin(e.g., where the R³ group is a succinyl, a methyl succinyl, or amyristoyl group) or an endoproteinase Lys-C.

These methods can be utilized with a sample from any source. In someembodiments, the sample is a purified preparation of the enzyme. Inother embodiments, the sample is an extract of a cell, tissue or organof a multicellular eukaryote, for example a mammal. The sample cancomprise, for example, a living cell or a homogenized mixture fromtissue.

These methods are not limited to detection of any class of enzyme,provided the enzyme is capable of removing the R³ moiety from thecompound. In some embodiments, the enzyme is a histone deacetylase(HDAC), for example a sirtuin, e.g., SIRT5.

An example of a compound useful for these methods is

More specific examples include

In some embodiments of these methods, the enzyme is quantified in thesample by measuring the rate of removal of the R³ moiety and comparingthat rate to the rate of removal of a known amount of the enzyme (i.e.,comparing to a standard curve of the enzyme action on the compound). Invarious embodiments, the rate of removal is determined by the increasedsignal from SIG after addition of a peptidase, and comparing theincreased signal to a standard curve that provides the quantity of theenzyme that leads to the increased signal.

The above-described compounds can also be used to identify an inhibitorfor an enzyme that removes the R³ moiety from any of the above-describedcompounds, by determining whether a putative inhibitor prevents removalof the R³ moiety from the compound. Thus, the invention also is directedto a method of determining whether a molecule inhibits an enzyme thatremoves a moiety from an ε-amino of a lysine, where the moiety is asuccinyl, a methyl succinyl, a formyl, or a myristoyl. The methodcomprises

(a) combining the enzyme and the molecule with any of theabove-described compounds to make an enzyme-molecule-compound mixture,where R³ of the compound is the moiety;

(b) incubating the enzyme-molecule-compound mixture under conditions andfor a time sufficient for the enzyme to remove the moiety in the absenceof the molecule; and

(c) determining whether the R³ is removed from the compound to anequivalent degree that R³ would be removed from the compound in theabsence of the molecule. In these methods, the failure of the removal ofR³ from the compound to an equivalent degree as in the absence of themolecule indicates that the molecule is an inhibitor of the enzyme.

The determination of whether the R³ moiety is removed can be by anymeans known in the art, for example by mass spectroscopy, an immunoassaywith an antibody that can distinguish between the compound with the R³group and without it, or an appropriate chromatographic method. In someembodiments, the compound is a substrate for a peptidase after theenzyme cleaves R³ from the compound but not if the R³ is not removedfrom the compound. In such as case, the determining step furthercomprises

(i) adding the peptidase to the mixture for a time sufficient for thepeptidase to cleave the resulting molecule between the R¹ and the COmoieties, such that SIG generates an increased signal relative to thesignal generated with the compound; and

(ii) determining whether SIG generates an increased signal relative tothe signal generated with the compound. Here, an increased signalindicates the removal of R³ from the compound.

These methods are not narrowly limited to the use of any particularpeptidase. In various embodiments, the peptidase is a trypsin (e.g.,where the R³ group is a succinyl, a methyl succinyl, or a myristoylgroup) or an endoproteinase Lys-C.

Further, these methods are not limited to detection of any class ofenzyme, provided the enzyme is capable of removing the R³ moiety fromthe compound. In some embodiments, the enzyme is a histone deacetylase(HDAC), for example a sirtuin, e.g., SIRT5.

An example of a compound useful for these methods is

More specific examples include

These methods can utilize high-throughput and automation methods andequipment known in the art to assay more than one moleculesimultaneously. Such methods are useful for assaying any number ofcompounds, for example from a chemical library. In some embodiments,more than 10 molecules are subjected to the method simultaneously. Inother embodiments, more than 50 molecules are subjected to the methodsimultaneously.

Preferred embodiments are described in the following example. Otherembodiments within the scope of the claims herein will be apparent toone skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by theclaims, which follow the examples.

Example. Compounds and Methods for Detecting SIRT5

The compound, N—(N^(α)-acetyl-L-Lysine(N^(ε)-succinyl))-AMC(Ac-Lys(Succ.)-AMC; AMC=7-amino-4-methylcoumarin) is depicted asCompound 1 and was synthesized as follows. Succinic anhydride (0.2 mmol,0.020 g) was added under an argon atmosphere to a suspension ofAc-Lys-AMC (0.2 mmol, 0.069 g) in DMF (1.3 ml). The mixture was stirredfor four hours at room temperature. All volatility was evaporated undervacuum. The residue was purified by flash chromatography (silica gel; 5%to 15% MeOH in CH₂Cl₂) to give a yellowish solid as an impure compound.The mixture was further purified by reverse phase chromatography (C18silica gel, 100% H₂O to 15% H₂O in MeOH to 100% MeOH) to afford 0.077 gof Ac-Lys(Succ.)-AMC as an off-white pure solid (purity >98% TLC, R_(f):0.45, 20% MeOH/CH₂Cl₂, 86% yield); C₂₂H₂₇N₃O₇, FW: 445.50.

The ¹H-NMR spectrum and mass spectroscopy analysis (MS:468.1 (M+Na⁺))were consistent with the structure depicted as Compound 1,Ac-Lys(Succ.)-AMC.

The compound, N,N′-Bis(α-acetyl-Lysine(ε-succinyl))-rhodamine 110((Ac-K(Succ.)₂-R110) was prepared as follows. Pyridine (1 ml) and1-ethyl-(3-dimethylaminopropyl)-carbodiimide hydrochloride (1.4 mmol,0.268 g, Chem-Impex-International, Cat. #00050) was added to a solutionof Rhodamine Green 560 chloride (rhodamine 110, 0.5 mmol, 0.183 g) andAc-Lys(Boc)-OH (1.05 mmol, 0.303 g, Bachem Cat. # E1040) in DMF (1 ml)at 0° C. under an argon atmosphere. The mixture was stirred at roomtemperature overnight. All volatility was evaporated by vacuum. Theresidue was purified by flash chromatography (silica gel; 5% to 15% MeOHin CH₂Cl₂) to yield a yellowish, semisolid impure intermediate (0.24 g,28% yield). The intermediate (0.24 g, 0.27 mmol) was dissolved in CH₂Cl₂(1.5 ml). Trifluoroacetic acid (1.5 ml) was added to this solution at 0°C. The mixture was stirred at room temperature for 1 hour. All thesolvents were evaporated by rotary evaporator to give 0.20 g of thedeprotected (deBOC) compound. Succinic anhydride (0.075 g, 0.075 mmol)was added to a suspension of the deBOC compound (0.10 g, 0.15 mmol) inDMF (1 ml). The resulting mixture was stirred overnight under an argonatmosphere at room temperature. All volatility was evaporated and themixture was purified by flash chromatography (10% to 30% MeOH in CH₂Cl₂)to yield a yellow, solid mixture. The mixture was purified again byreverse phase chromatography (C18 silica gel, 100% H₂O to 10% H₂O inMeOH to 100% MeOH) to afford 0.022 g of pure compound: C₄₄H₅₀N₆O₁₃, FW:870.90. The NMR spectrum was consistent with the structure depicted asCompound 2, N, N′-Bis(α-acetyl-Lysine(ε-succinyl))-rhodamine 110((Ac-K(Succ.))₂-R110).

The compound N—(N^(α)-acetyl-L-Lysine(N^(ε)-formyl))-AMC(Ac-Lys(Form.)-AMC) is depicted as Compound 3 and was synthesized asfollows: Acetic anhydride (0.06 ml, Aldrich) was added to a suspensionof Ac-Lys-AMC (0.0143 g, 0.041 mmol, Bachem 1-1040) in dry formic acid(0.15 ml, treated by 3 Å molecular sieves) slowly with stirring over aperiod of 7 minutes under an argon atmosphere at 0° C. The mixture wasstirred for two hours at room temperature and was then quenched intoether (50 ml). A precipitate was formed and the precipitate was furtherwashed by ether. After filtering off the solution, the mixture waspurified by reverse phase chromatography (C18 silica gel, 100% H₂O to100% MeOH) to afford 0.010 g of impure compound as an off-white solid.The solid was crystallized from MeOH to afford 0.0085 g ofAc-Lys-N^(ε)-Formyl-AMC as a white solid (R_(f): 0.10, 50% MeOH/CH₂Cl₂,55% yield), C₁₉H₂₃N₃O₅, FW: 373.40. The NMR spectrum was consistent withthe structure depicted as Compound 3.

The compound, N—(N^(α)-acetyl-L-Lysine(N^(ε)-myristoyl))-AMC,Ac-Lys(Myr.)-AMC is depicted as Compound 4 and was prepared as follows.Myristoyl chloride (0.024 g, 0.010 mmol) was added to a suspension ofAc-Lys-AMC (0.022 g, 0.065 mmol) in DMF (1.0 ml) and pyridine (0.2 ml)slowly with stirring over a period of 5 minutes under argon atmosphereat 0° C. The mixture was stirred overnight at room temperature. Afterall volatility was evaporated under vacuum, the residue was purified byflash chromatography (silica gel, from 1% methanol in methylene chlorideup to 20% methylene chloride) to afford 0.016 g of pure compound as awhite solid. (R_(f): 0.48, 10% MeOH/CH₂Cl₂, 55% yield), C₄₄H₅₀N₆O₁₃, FW:555.75. The NMR spectrum was consistent with the structure depicted asCompound 4.

The compound, N—(N^(α)-acetyl-L-Lysine(N^(ε)-methyl succinyl))-AMC(Ac-Lys(Methyl Succ.)-AMC) is depicted as Compound 5 and was prepared asfollows. To a suspension of Ac-AMC-Succinyl-Lys (0.018 g, 0.040 mmol) inether (0.3 ml) was added freshly made diazomethane (2 ml) under an argonatmosphere at 0° C. The mixture was stirred for two hours at roomtemperature. All volatility was evaporated and diazomethane (2 ml) wasadded again. The mixture was stirred another one hour at roomtemperature. After all volatility was evaporated, the residue waspurified by flash chromatography (MeOH in methylene chloride from 5% to20%) to give Ac-Lys(Methyl Succ.)-AMC (0.015 g, 82% yield) as a whitesolid (R_(f): 0.31, 20% MeOH/CH₂Cl₂), C₂₃H₂₉N₃O₇, FW: 459.49. The NMRspectrum was consistent with the structure depicted as Compound 5.

As noted in the Background of the Invention section above, the proteasetrypsin catalyzes the hydrolytic cleavage of amide bonds on the carboxylside of unmodified lysine residues. Hydrolytic cleavage of lysyl amidebonds with AMC or rhodamine 110 (dye components of Compounds 1 and 2,respectively) releases the free amino forms of the dyes. This elicits anupward shift of the dye's fluorescence emission peak wavelength(bathochromic shift), for example to 460 nm for AMC or 530 nm forrhodamine 110. If succinylation of the ε-amino function of the lysinemoieties in compounds such as 1 and 2 renders the amide bond between thecarbonyl of lysine and the amino of dye groups such as AMC or rhodamineresistant to trypsin cleavage then, upon desuccinylation, the lysine/dyeamide bond would become cleavable by trypsin. Note that removal of asingle succinyl group from 2, followed by cleavage of the desuccinylatedlysine would yield a compound with a single free amino function,Ac-K(Succ.)-R110. Monoamides of rhodamine 110 also have increasedfluorescence emission at 530 nm relative to 2, although they are lessfluorescent than rhodamine 110 itself (Liu et al., 1999). Thus,monitoring the fluorescence at the peak wavelength of the amino form ofthe dye would allow the desuccinylation reaction to be quantified. Aschematic for such an assay is depicted for Compound 1 and SIRT5 inFIG. 1. The efficacy of trypsin for use in the second step of such anassay is demonstrated by the data presented in FIG. 2. Trypsin treatment(2 mg/ml) elicits an immediate increase in the fluorescence of asolution of Ac-Lys-AMC (Compound 6—FIG. 1) at the excitation andemission wavelengths of free AMC (Ex. 360 nm; Em. 460 nm), with maximumfluorescence achieved in 2 min. or less and ˜⅔ of that maximum occurringin 1 min. (FIG. 2). In contrast, when Ac-Lys(Succ.)-AMC (1) is subjectedto the same trypsin treatment, no change in fluorescence is observedover the entire course of the fluorescence measurement.

As demonstrated above for Compound 1, succinylation of the ε-aminofunction of lysine confers complete resistance to the trypsin cleavageand release of the free dye group, which occurs in the otherwiseidentical, but non-succinylated Compound 6 (FIG. 1). Therefore, if SIRT5is capable of catalyzing the desuccinylation of a compound such as 1, itshould be possible to perform a desuccinylation assay by the two-stepprocedure depicted in FIG. 1. However, SIRT5 desuccinylation activitywith a single-lysine substrate such as compound 1 is by no means acertainty. While the activity of SIRT5 with the acetylated four residuepeptide lysine substrate, Ac-Arg-His-Lys-Lys(ε-acetyl)-AMC isexceedingly weak (Nakagawa, T. et al., 2009), its activity with thesingle-lysine acetylated substrate, Ac-Lys(ε-acetyl)-AMC, isconsiderably worse (FIG. 5 and Table 2, further discussed below).Genuine SIRT5 desuccinylation activity with Ac-Lys(ε-succinyl)-AMCshould of course depend on the presence of SIRT5, but also on thepresence of the sirtuin co-substrate, NAD⁺. A trial SIRT5 assay wasconducted with Ac-Lys(ε-succinyl)-AMC under high enzyme concentrationconditions (5 μg SIRT5 per 50 μl reaction) necessary for detection ofSIRT5 deacetylation activity with Ac-Arg-His-Lys-Lys(ε-acetyl)-AMC (FIG.3). Desuccinylation of Ac-Lys(ε-succinyl)-AMC was not only entirelydependent on the presence of SIRT5 and NAD⁺ but was, moreover, at least250-fold greater than the deacetylation ofAc-Arg-His-Lys-Lys(ε-acetyl)-AMC.

As seen in FIG. 3, SIRT5 desuccinylated Ac-Lys(Succ.)-AMC far moreefficiently than it deacetylated Ac-Arg-His-Lys-Lys(ε-acetyl)-AMC, asubstrate which had, heretofore, been the most effective SIRT5fluorogenic substrate known (U.S. Patent Application Publication20060014705; Schlicker et al., 2008). In the FIG. 3 assays, 5 μg ofSIRT5 (3 μM in 50 μl), 50 μM peptide substrate and a 60 min. incubationwere used in order to achieve significant deacetylation ofAc-Arg-His-Lys-Lys(ε-acetyl)-AMC. Under these conditions nearly 50% ofthe Ac-Lys(Succ.)-AMC had been desuccinylated, so theSIRT5/NAD⁺-dependent fluorescence increase would significantlyunderestimate the initial desuccinylation rate and the kinetic capacityof SIRT5 with Ac-Lys(Succ.)-AMC substrate. When assayed with lowerquantities of SIRT5, lower Ac-Lys(Succ.)-AMC concentrations. and ashorter incubation time, statistically significant desuccinylation isachieved with 5 ng SIRT5/50 μl (3 nM), 2 or 10 μM Ac-Lys(Succ.)-AMC anda 20 min. incubation (FIG. 4). As noted in the Background of theInvention section, an assay's lower limit for determination of aninhibitor's IC₅₀ is one-half the enzyme concentration. Thus, an assaybased on Ac-Lys(Succ.)-AMC, as opposed to an acetylated fluorogenicsubstrate, enables at least a three orders of magnitude improvement inthe ability to detect or characterize high-potency inhibitors.

SIRT5 initial rate desuccinylation kinetics were determined as afunction of the concentration of Ac-Lys(Succ.)-AMC and were compared inthis regard to the equivalent single-lysine acetylated substrate,Ac-Lys(ε-acetyl)-AMC (FIG. 5, Ac-Lys(Ac)-AMC). Aside from the vastlygreater initial rates achieved with the succinylated substrate, it isnotable that the Ac-Lys(Succ.)-AMC substrate displays saturationkinetics, allowing the determination of the Michaelis-Menten constantsK_(m) and V_(max) (K_(m)=108 μM; V_(max)=490 pmol/min/μg; FIG. 5A),whereas the rate dependence on the concentration of Ac-Lys(Ac)-AMCremains linear over the concentration range of the assay (FIG. 5B). Thisresult with Ac-Lys(Ac)-AMC is similar to the high and uncertain K_(m)estimate for the substrate Ac-Arg-His-Lys-Lys(ε-acetyl)-AMC (8.9 mM inassay with a maximum substrate concentration of 5 mM—U.S. PatentApplication Publication 20060014705) and is consistent with a greaterSIRT5 binding affinity for substrates that are succinylated rather thanacetylated on the ε-amino function of lysine. The ability to obtainenzyme kinetic constants with the substrate(s) and assay of the presentinvention has utility for SIRT5 research and related drug discoveryefforts, enabling for example the calculation of intrinsic inhibitorconstants (Kis) as opposed to relative, assay-dependent constants suchas the IC₅₀.

SIRT5 kinetic parameters were also obtained for desuccinylation of aconstant concentration of Ac-Lys(Succ.)-AMC (0.5 mM) as a function ofthe concentration of the cosubstrate NAD⁺ (FIG. 6). TheSIRT5/Ac-Lys(Succ.)-AMC kinetic parameters k_(cat) and k_(cat)/K_(m)were calculated from the data of FIG. 5 and the NAD⁺ kinetic data ofFIG. 6. These are listed in Table 1 along with literature values forhuman recombinant sirtuins with unlabeled acetylated lysine peptidesubstrates (histone H3 residues 1-20 or 4-15 acetylated on lysine-9(“K9Ac”)). It is notable that the k_(cat) and k_(cat)/K_(m) values forSIRT5 with Ac-Lys(Succ.)-AMC are similar to or in some cases greaterthan those of the bona fide sirtuin deacetylases SIRTs 1-3 withunlabeled acetylated peptide substrates.

TABLE 1 Kinetic Parameters of Recombinant Human Sirtuins: SIRT5 withAc-Lys(Succ.)-AMC In Comparison to SIRTs 1-3, 5 with Acetylated PeptideSubstrates K_(m) Acetylated or (Lysyl K_(m) Succinylated Variedsubstrate) (NAD⁺) k_(cat) k_(cat)/K_(m) Enzyme Lysyl Substrate SubstrateμM μM s⁻¹ s⁻¹M⁻¹ SIRT5 ^(a) Ac-Lys(Succ.)-AMC Lysyl 108 0.26 2450 SIRT5^(a) Ac-Lys(Succ.)-AMC NAD⁺ 360 0.29 798 SIRT1 ^(b) Hist. H3 4-15, K9AcNAD⁺ 80 0.079 988 SIRT2 ^(b) Hist. H3 4-15, K9Ac NAD⁺ 46 0.021 457 SIRT2^(c) Hist. H3 1-20, K9Ac Lysyl 24 0.24 10,000 SIRT3 ^(b) Hist. H3 4-15,K9Ac NAD⁺ 118 0.009 76 SIRT5 ^(b) Hist. H3 4-15, K9Ac NAD⁺ 861 0.003 3.5^(a) Data from present work, calculated from K_(m) and V_(max) values ofFIGS. 7 and 8. ^(b) Data from Du et al., 2009. ^(c) Data from Borra etal., 2004.

A particularly important application of the present invention lies inthe screening for and characterization of modulators (inhibitors oractivators) of SIRT5 activity. The substrates provided may be used inhomogenous assays. That is, the assays may be performed by a simpleprocess of successive solution addition and mixing steps in a singlevessel, for example the well of a microplate. As such, the assay may beeasily adapted to automated liquid handling equipment and low-volumevessels (e.g. 96, 384 & 1536 well microplates) used, for example, inhigh-throughput screening of chemical libraries for modulators. HighSIRT5 assay sensitivity is provided by the combination of the enzymekinetic characteristics of the fluorophore-labeled succinylated-lysinesubstrates (FIGS. 3-6) and structures which allow trypsin-drivendesuccinylation-dependent fluorescence increases (FIGS. 1 and 2). Asnoted earlier, this high sensitivity allows the use of low enzymeconcentrations (e.g. 3 nM) in SIRT5 assays. Low enzyme concentrationsare advantageous both for minimizing the costs of high-throughputscreening and allowing the identification and characterization ofhigh-potency modulators. To demonstrate the utility of theAc-Lys(Succ.)-AMC substrate in characterizing SIRT5 inhibitors, twoknown SIRT5 inhibitors were chosen, suramin (U.S. Patent ApplicationPublication 200600147050; Schuetz et al., 2007) and the general sirtuininhibitor and reaction product nicotinamide. The data and calculationsfor determining their IC₅₀'s is shown in FIGS. 7 and 8. The suramin IC₅₀obtained (27.3 μM; FIG. 7) agrees well with the value of 22 μM obtainedin a radioactive assay with chemically acetylated chicken histones(Schuetz et al., 2007). The nicotinamide IC₅₀, under the sameconditions, was 29.0 μM (FIG. 8). Nicotinamide is presumed to be a SIRT5inhibitor, since, as is the case for other sirtuins, SIRT5 hasnicotinamide-NAD⁺ exchange activity (Schuetz et al., 2007). However, toour knowledge, no detailed characterization of SIRT5 inhibition bynicotinamide has been reported in the literature.

Since desuccinylation activity had not previously been attributed to anysirtuin (class III HDACs, i.e. NAD⁺-dependent lysine deacetylases) or toany of the other, hydrolytic lysine deacetylases (class I, II and IVHDACs), it was investigated whether such activity was unique to SIRT5and whether a substrate such as Compound 1 might form the basis of aSIRT5-specific assay. The Ac-Lys(Succ.)-AMC substrate was tested foractivity with recombinant preparations of human HDACs 1-11, SIRTs 1-4, 6and 7, a complex of HDAC3 with a fragment of the activating proteinNCOR1 and with HeLa nuclear extract, a rich source of active HDACs intheir native multiprotein complexes. No other sirtuin showed anyactivity with Ac-Lys(Succ.)-AMC (50 μM Ac-Lys(Succ.)-AMC, 500 μM NAD⁺;Table 2). Extremely low, but detectable activities (Table 2; FIG. 9)were found for two class I HDACs (HDAC2 and HDAC3/NCOR1 complex) and forHeLa nuclear extract (ENZO Life Sciences Cat. # BML-KI142), apreparation rich in the class I HDACs 1, 2 and 3. For comparison, Table2 includes the activities of all HDACs and SIRTs with the single-lysinesubstrate Ac-Lys(Ac)-AMC. The use of a longer peptide or otherwisemodified (e.g., Lys(ε-trifluoroacetyl)) substrates, rather thanAc-Lys(Ac)-AMC, improves the activity of a number of HDACs and SIRTs.Examples of these activities are also included for comparison as “OtherSubstrates” (Table 2).

TABLE 2 Activities of Recombinant Human HDACs and Sirtuins withAc-Lys(Succ.)- AMC, Ac-Lys(Ac.)-AMC and other Fluorogenic SubstratesActivity with Activity with Ac-Lys(Succinyl)-AMC Ac-Lys(Acetyl)-AMCOther Substrates/Activity Enzyme (pmol/min/μg)^(a) (pmol/min/μg)^(b)(pmol/min/μg)^(c) HDAC1 Undetectable 14.8 Ac-RHKK(Ac)-AMC/21 (0.5 μg, 60min.) HDAC2 0.28 554 (Ac-Lys(Ac))₂-R110/502 HDAC3 Undetectable 3.8 (0.5μg, 60 min.) HDAC3/NCOR1 0.40 668 Ac-RHKK(Ac)-AMC/687 Complex HDAC4Undetectable Undetectable Ac-LGK(TFAc)-AMC/1390 (2 μg, 60 min) (2 μg, 60min) HDAC5 Undetectable Undetectable Ac-LGK(TFAc)-AMC/9960 (2 μg, 60min) (2 μg, 60 min) HDAC6 Undetectable 1.0 (0.5 μg, 60 min.) HDAC7Undetectable Undetectable Ac-LGK(TFAc)-AMC/1670 (2 μg, 60 min) (2 μg, 60min) HDAC8 Undetectable 0.091 Ac-RHK(Ac)K(Ac) (0.5 μg, 60 min) AMC/4.5HDAC9 Undetectable 0.027 Ac-LGK(TFAc)-AMC/3330 (2 μg, 60 min) HDAC10Undetectable 1.21 Ac-RHKK(Ac)-AMC/2.72 (0.3 μg, 60 min) HDAC11Undetectable 4.15 Ac-RHKK(Ac)-AMC/6.09 (0.3 μg, 60 min) SIRT1Undetectable 0.391 Ac-RHKK(Ac)-AMC/64 (1 μg, 60 min) SIRT2 Undetectable0.016 Ac-QPKK(Ac)-AMC/18.6 (2.6 μg, 60 min) SIRT3 Undetectable 0.125Ac-QPKK(Ac)-AMC/10.8 (3 μg, 60 min) SIRT4 Undetectable Undetectable (3μg, 60 min) (3 μg, 60 min) SIRT5 115    0.0174 Ac-RHKK(Ac)-AMC/0.364SIRT6 Undetectable Undetectable Ac-RHKK(Ac)-AMC/0.052 (3 μg, 60 min) (3μg, 60 min) SIRT7 Undetectable Undetectable (3 μg, 60 min) (3 μg, 60min) HeLa Nuclear Extract  0.012 31 ^(a)Activities determined with 50 μMAc-Lys(Succ.)-AMC (HDACs and SIRTs) with 500 μM NAD⁺ added for SIRTs.^(b)Activities determined with 50 μM Ac-Lys(Ac.)-AMC (HDACs and SIRTs)with 500 μM NAD⁺ added for SIRTs. ^(c)Activities determined with 50 μMof indicated peptide substrates, single-letter amino acid code (HDACsand SIRTs) with 500 μM NAD⁺ added for SIRTs. Substrate for the class IIaHDACs (4, 5, 7, 9) has trifluoroacetyl (TFAc) rather than acetylfunction on the ε-amino group of lysine (Bradner, J. E. et al. NatureChem. Biol. 6, 238-243 (2010)). The substrate Ac-Lys(Ac))₂-R110 (EnzoLife Science Cat. # BML-KI572, a component of Fluor de Lys-Green HDACAssay Kit, Cat. #BML-AK530) is an analog of 2, but bears acetyl ratherthan succinyl functions.

The data of Table 2 and FIG. 9 indicate that, among human HDACs andSIRTs, desuccinylation activity with the substrate Ac-Lys(Succ.)-AMC(Compound 1) is nearly completely specific to SIRT5. Minor activity withclass I HDACs could, however, contribute to a non-SIRT5 background inthe context of assays on intact cells or tissues, or cell or tissueextracts. Factors which could increase this background relative to SIRT5activity include: 1) greater relative expression levels of the class IHDACs, 2) enhancement of class I HDAC activity when part of nativemultiprotein complexes (note the measurable activity of HDAC3/NCOR1 asopposed to the absence of activity from HDAC3), and 3) for intact cellsand tissues, relative inaccessibility of SIRT5 to the substrate (nativeSIRT5 resides inside the mitochondrial inner membrane). The first two ofthese factors could readily be overcome by inclusion of an inhibitor,such as trichostatin A, which inhibits class I HDACs but not SIRTs(Bhalla et al., 2005).

An assay employing Ac-Lys(Ac.)-AMC to measure the HDAC and sirtuindeacetylase activity of intact cultured cells has been described(Howitz, K. T. et al. Nature 425, 191-196 (2003); Product Manual of EnzoLife Sciences Cat. # BML-AK-503, “HDAC Fluorimetric Cellular ActivityAssay Kit”, Appendix F). Ac-Lys(Ac)-AMC is added to the culture medium,enters the cells and is deacetylated by HDACs and sirtuins in theirnative intracellular context. Measurement of the amount of Ac-Lys-AMCproduced by intracellular deacetylation is then accomplished bydetergent lysis of the cells and release of the AMC fluorophore bytrypsin. We therefore investigated whether Ac-Lys(Succ.)-AMC could beused to measure intracellular desuccinylation activity, in a similarfashion, with cultured HeLa cells. However, as can be seen from FIG.12A, no significant desuccinylation occurs after HeLa cells have beencultured four hours in the presence of 200 μM Ac-Lys(Succ.)-AMC. Incontrast, a parallel set of HeLa cell treatments, with 200 μMAc-Lys(Ac.)-AMC (FIG. 12B), produces significant deacetylation in fourhours, the vast majority of which (>90%) is sensitive to the class I/IIHDAC inhibitor trichostatin A (TSA). These latter, deacetylation resultsare consistent with previous observations made with this assay system(see Howitz, K. T. et al. Nature 425, 191-196 (2003) and Appendix F).

Aside from the far more limited range of enzymes capable ofdesuccinylating Ac-Lys(Succ.)-AMC, as opposed to deacetylatingAc-Lys(Ac.)-AMC (Table 2), another factor may be contributing to thelack of an intracellular desuccinylation signal (FIG. 10), namely thetwo membrane barriers (plasma and inner-mitochondrial membranes)standing between the medium and SIRT5. Absent a specific membranetransport protein, a molecule's membrane permeability (i.e. capacity todiffuse across the lipid bilayer) generally goes down with increasingsize and with increasing polarity or charge (Stein, 1986). SinceAc-Lys(Succ.)-AMC is both larger than Ac-Lys(Ac.)-AMC (MW 445.5 vs.387.4) and carries a negative charge, it is probable that its lowermembrane permeability presents a greater kinetic barrier to diffusionacross both the plasma and inner-mitochondrial membranes. Moreover,since Ac-Lys(Succ.)-AMC is negatively charged and since there is anegative inside membrane potential maintained across both of thesemembranes, the internal equilibrium concentration of the anionicAc-Lys(Succ.)-AMC would likely be significantly lower than its externalconcentration (Johnson et al., 1981). This would in turn impose akinetic constraint on a desuccinylating enzyme such as SIRT5.

In order to circumvent the problems likely imposed by these membranebarriers, whole HeLa cell lysates were prepared and tested forAc-Lys(Succ.)-AMC desuccinylating activity (FIG. 11). Since the cellswere lysed, the internal pool of the SIRT5 co-substrate NAD⁺ was dilutedinto a much larger volume, allowing the effects of NAD⁺'s presence orabsence to be tested. It was also possible to use the membraneimpermeant SIRT5 inhibitor suramin instead of nicotinamide. This has theadvantage of avoiding possible complications resulting from metabolismof nicotinamide to NAD⁺. Generalizations that can be drawn from thisdata include: 1) increases over the 2 hr. are dependent on the lysatesince NL(No Lysate) fluorescences were approximately equal to the 0 hr.samples; 2) the maximum fluorescence increase (22,000) over 0 hr./NLoccurs in the absence of the class I/II HDAC inhibitor and the presenceof NAD⁺, indicating contributions both from sirtuins (probably SIRT5)and non-sirtuin HDACs (probably class I); 3) consistent with point 2),the addition of TSA and the addition of suramin to the +NAD⁺ conditioneach produce ˜50% inhibition of the total 2 hr. fluorescence increase.This latter point is particularly interesting in that it suggests,despite the relative poor in vitro activity of the recombinant class IHDACs, that they may contribute levels of total cellularlysine-desuccinylating activity roughly comparable to those of SIRT5.Further, since the class I HDACs are primarily nuclear enzymes, itsuggests that a proteomic investigation into the possible presence oflysine-succinylated proteins in that compartment may be warranted.Moreover, the apparent specificity, within the non-sirtuin HDACs, of theAc-Lys(Succ.)-AMC substrate for the class I HDACs might provideinformation useful in the design of class I-specific inhibitors.

The results of the cell extract assays (FIG. 11) and SIRT5 kineticstudies suggest assay conditions for the measurement of SIRT5-specificdesuccinylating activity in cell or tissue extracts or other mixedprotein preparations (e.g. subcellular fractions such as isolatedorganelles, or partially purified cellular proteins such aschromatographic fractions). These conditions would simply be todetermine the time-dependent increase in fluorescence (after trypsinrelease of AMC) in the presence of TSA (1 μM or higher),Ac-Lys(Succ.)-AMC (concentrations from 5 μM to 500 μM would be feasible)and NAD⁺ (concentrations from 30 μM to 3 mM would be feasible). Asuitable control for such a measurement would be to also perform theassay in the absence of NAD⁺. Note that in the presence of TSA, thewithdrawal of NAD⁺ eliminates the 0 to 2 hr. fluorescence increase(actually turns slightly negative but within the variability of the 0hr. and No Lysate samples), thus confirming that the increase resultsfrom SIRT5 activity (compare fifth and second bars in FIG. 11B). Analternative means to determine the SIRT5 desuccinylation activity in acell or tissue extract would be determine the total desuccinylationsignal in the presence of Ac-Lys(Succ.)-AMC and NAD⁺ as above, butwithout TSA. The SIRT5 activity would then equal the decrease in of thistime-dependent fluorescence change in the presence of a SIRT5 inhibitor.The inhibitor could be suramin (200 μM or higher) or nicotinamide (200μM or higher).

The cell extract experiments of FIG. 11 were performed with 50 μMAc-Lys(Succ.)-AMC and 500 μM NAD⁺ (when present). These conditions werechosen to be the same as those of the survey of recombinant sirtuins andHDACs (Table 2) and, in the case of the 50 μM Ac-Lys(Succ.)-AMC, tominimize background fluorescence due to the substrate. Under theseconditions, as noted above, TSA-sensitive activity (class I HDACs)contributed about half of the total desuccinylation in the absence ofinhibitors. However, it should be possible to increase the SIRT5 portionof the total signal, particularly by increasing the NAD⁺ concentration.Taking the K_(m) for NAD⁺ as 360 μM (FIG. 6), the Michaelis-Mentenequation yields a result of 58% of V_(max) at 500 μM. Increasing theNAD⁺ concentration to 5 mM, for example, would increase the SIRT5 rateto 93% of V_(max), while having no effect on the non-SIRT5 activity. TheK_(m)'s of class I HDACs for Ac-Lys(Succ.)-AMC have not been determined,but given their low activity with this substrate it is likely that theyare at least as high as that of SIRT5 (108 μM). Given that, below K_(m),the enzyme velocity is roughly linear with substrate concentrations, theAc-Lys(Succ.)-AMC could be lowered from 50 μM, thereby decreasingfluorescence background, while not decreasing the ratio of SIRT5 toclass I HDAC activity.

It was possible with the HeLa lysate system to measure apparent SIRT5Ac-Lys(Succ.)-AMC desuccinylating activity (FIG. 11) whereas this wasnot the case with intact HeLa cells (FIG. 10). Given that the cellnumber used in both assays were similar (equivalent of 28×10⁴ for thecell extract vs. seeding at 4×10⁴ per well and growth for ˜20 hr. forthe intact cells) and that the intact cell incubation was twice as longas that for the cell extracts, the membrane permeability factorsdiscussed above are the most likely cause for this discrepancy. Chemicalmodifications to the Ac-Lys(Succ.)-AMC that render the distalcarboxylate of the succinyl group neutral, rather than negative atneutral pH, could enable an intact cell assay that is capable of SIRT5detection. Such modifications might include amidation andesterification. Acetoxymethyl (AM) esterification, which tends to beremoved intracellularly by non-specific esterases, could improve themembrane permeability, while also allowing for restoration of theoriginal substrate molecule within the cell.

Another example of a fluorogenic substrate for assay of SIRT5desuccinylase activity is Compound 2 ((Ac-Lys(Succ.))₂-R110). The SIRT5and NAD⁺-dependent increase in fluorescence was determined for(Ac-Lys(Succ.))₂-R110 in comparison to that for deacetylase substrate(Ac-Lys(Ac.))₂-R110 (Fluor de Lys®-Green, Enzo Life Sciences Cat. #KI-572) and the data are shown in FIG. 12.

As was the case for the Ac-Lys(Succ.)-AMC (FIG. 3), the(Ac-Lys(Succ.))₂-R110 substrate is desuccinylated by SIRT5 in a time andNAD⁺-dependent manner (FIG. 12). Further, as was the case withAc-Lys(Succ.)-AMC in comparison to Ac-Lys(Ac.)-AMC (Table 2), SIRT5operates far more efficiently with the succinylated substrate.

Various HDAC enzymes, including SIRT1, 3, 6 and 7, were tested foractivity with Compounds 4 (Ac-Lys(Myr.)-AMC) and 5 (Ac-Lys(MethylSucc.)-AMC) to determine the lysyl-N^(ε) de-myristoyl (Compound 4) andde-methylsuccinyl (Compound 5) activity of those enzymes. Results areshown in Table 3. As shown therein, some of those enzymes had activitywith those reagents, although the activity was much lower than theactivity with Ac-Lys(Acetyl)-AMC.

TABLE 3 Activities of Recombinant Human HDACs and Sirtuins withAc-Lys(Myristoyl)-AMC, Ac-Lys(Methyl Succinyl)-AMC, Ac-Lys(Ac.)-AMCActivity with Activity with Activity with Ac-Lys(Myristoyl)-Ac-Lys(Methyl Ac-Lys(Acetyl)- AMC Succinyl)-AMC AMC Enzyme(pmol/min/μg)^(a) (pmol/min/μg)^(a) (pmol/min/μg)^(b) HDAC1 1.651 0.79814.8 HDAC2 83.05 31.330 554 HDAC3 0.963 0.431 3.80 HDAC3/ 79.023 53.750668 NCOR1 Complex HDAC5 Undetectable Undetectable Undetectable (2 μg, 60min) (2 μg, 60 min) (2 μg, 60 min) HDAC7 Undetectable UndetectableUndetectable (2 μg, 60 min) (2 μg, 60 min) (2 μg, 60 min) HDAC8Undetectable 0.003 0.091 (0.5 μg, 60 min) HDAC9 Undetectable 0.135 0.027(2 μg, 60 min) SIRT1 0.043 0.002 0.391 SIRT3 0.012 0.002 0.125 SIRT60.074 0.006 0.436 SIRT7 Undetectable Undetectable Undetectable (3 μg, 60min) (3 μg, 60 min) (3 μg, 60 min) HeLa Nuclear 0.819 1.764 31 Extract^(a)Activities determined with 50 μM Ac-Lys(Succ.)-AMC (HDACs and SIRTs)with 500 μM NAD⁺ addec for SIRTs. ^(b)Activities determined with 50 μMAc-Lys(Ac.)-AMC (HDACs and SIRTs) with 500 μM NAD⁺ added for SIRTs.Materials and Methods

Enzymes and Protein Extracts.

Enzyme and extract catalog products obtained from ENZO Life Sciencesincluded: HDAC1 (Cat. # BML-SE456), HDAC2 (Cat. # BML-SE500), HDAC3(Cat. # BML-SE507), HDAC3/NCOR1 Complex (Cat. # BML-SE515), HDAC6 (Cat.# BML-SE508), HDAC8 (Cat. # BML-SE145), HDAC10 (Cat. # BML-SE559),HDAC11 (Cat. # BML-SE560), SIRT1 (Cat. # BML-SE239), SIRT2 (Cat. #BML-SE251), SIRT3 (Cat. # BML-SE270), SIRT5 (Cat. # BML-SE555), HeLaNuclear Extract (Cat. # BML-KI140), Trypsin (“Fluor de Lys Developer”,80 mg/ml bovine trypsin in 1 mM HCl, Cat. # BML-KI105 or “Fluor de LysDeveloper II”, 80 mg/ml bovine trypsin in 1 mM HCl, Cat. # BML-KI176).Enzymes produced internally at Enzo Life Sciences International,Plymouth Meeting, Pa. included: HDAC4 (human recombinant, residues626-824 of GenBank accession # NP_006028 with an N-terminal His-tag(MGSSHHHHHHSSGLVPRGSHMAS, one-letter code), expressed in E. coli), HDAC5(human recombinant, residues 657-1123 of GenBank accession #NP_001015053 with an N-terminal His-tag (MGSSHHHHHHSSGLVPRGSHMAS,one-letter code), expressed in E. coli), HDAC7 (human recombinant,residues 483-903 of GenBank accession # NP_056216 with an N-terminalHis-tag (MGSSHHHHHHSSGLVPRGSHMAS, one-letter code), expressed in E.coli), HDAC9 (human recombinant, residues 644-1066 of GenBank accession# NP_848510 with an N-terminal His-tag (MGSSHHHHHHSSGLVPRGSHMAS,one-letter code), expressed in E. coli). Enzymes obtained from BPSBioscience, San Diego, Calif. included: human recombinants SIRT4 (Cat.#50015), SIRT6 (Cat. #50017), SIRT7 (Cat. #50018). HeLa whole cellextract was obtained from HeLa S3 cells (American Type CultureCollection) grown in a medium of MEM/10% fetal bovine serum (FBS) asfollows: 1) 9×10⁶ cells were suspended by trypsinization, followed by aphosphate buffered saline (PBS) rinse and addition of MEM/10% FBS (1.5mL) to eliminate trypsin before transfer to a 15 mL conical tube; 2)cells were sedimented by a low speed centrifugation, gently resuspendedin PBS and sedimented again and supernatant removed; 3) lysis wasinduced by resuspension in 0.8 mL 50 mM Tris/HCl, pH 8.0 with thenon-denaturing detergent NP-40 (0.5%), placement on ice and briefvortexing every 5 min. for 30 min.; 4) 100 μl of a concentrated saltsolution in the same buffer was added in order to bring the final buffercomposition to 50 mM Tris/C1, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mMMgCl₂, 0.5% NP-40; 5) particulate matter was cleared by centrifugationand the cleared lysate supernatant transferred to a 1.5 mL Eppendorftube and stored on ice until use in activity assays.

Reagents.

Enzyme assay reagents were components from either the “SIRT1Fluorometric Drug Discovery Kit” from Enzo Life Sciences (ELS) (Cat. #BML-AK555) or the “HDAC Fluorimetric Cellular Activity Assay Kit” (ELSCat. # BML-AK503) plus: “Fluor de Lys Substrate” (Ac-Lys(Ac.)-AMC; ELSCat. # BML-KI104), trichostatin A (TSA) (ELS Cat. # BML-GR309-9090),“Fluor de Lys Deacetylated Standard” (Ac-Lys-AMC; ELS Cat. # BML-KI142);“Fluor de Lys-SIRT2 Deacetylase Substrate” (Ac-Gln-Pro-Lys-Lys(Ac)-AMC;ELS Cat. # BML-KI179), “Fluor de Lys-HDAC8 Deacetylase Substrate”(Ac-Arg-His-Lys-Lys(Ac)-AMC; ELS Cat. # BML-KI178), “Fluor de Lys-GreenSubstrate” ((Ac-Lys(Ac))₂-R110; ELS Cat. # BML-KI572); the class IIaHDAC substrate Ac-Leu-Gly-Lys(TFAc)-AMC (Bradner et al., 2010)synthesized at Enzo Life Sciences International, Exeter, UK.

Activity Assays with Enzymes and Protein Extracts.

Assays were performed at 37° C., according to the manufacturer'sinstructions for the “SIRT1 Fluorometric Drug Discovery Kit” from EnzoLife Sciences (ELS) (Cat. # BML-AK555), with exceptions described asfollows. Where indicated in figures and text, SIRT1 enzyme was replacedwith another enzyme or extract and the “Fluor de Lys-SIRT1 Substrate”(Ac-Arg-His-Lys-Lys(Ac)-AMC; ELS Cat. # BML-KI-177) was replaced withanother acetylated, succinylated or trifluoroacetylated substrate. Inassays with non-sirtuin enzymes (class I HDACs 1, 2, 3 and 8; class IIenzymes HDACs 4-7, 9 and 10; class IV enzyme HDAC11) NAD⁺ was omittedand the nicotinamide in the “Developer” (trypsin) solution was replacedwith 1 μM TSA. In the HeLa cell extract experiments (FIG. 11), the“Developer” solutions were varied in order that the final inhibitorconcentrations for all samples were suramin (200 μM) and TSA (1 μM).

Activity Assays with Intact HeLa Cells.

Assays (FIG. 10) were performed according to the manufacturer'sinstructions for the ““HDAC Fluorometric Cellular Activity Assay Kit”(ELS Cat. # BML-AK503), with exceptions described as follows. Whereindicated the “Fluor de Lys Substrate” (Ac-Lys(Ac.)-AMC; ELS Cat. #BML-KI104) was replaced with Ac-Lys(Succ.)-AMC (1). The “Developer/CellLysis Buffer” solutions were varied in order that the final inhibitorconcentrations for all samples were nicotinamide (1 mM) and TSA (1 μM).

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In view of the above, it will be seen that several objectives of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

What is claimed is:
 1. A method of determining whether at least onemolecule inhibits an enzyme that removes a methyl succinyl moiety froman ε-amino of a lysine, the method comprising: (a) mixing the enzyme anda molecule with a compound having the formula

wherein SIG is a fluorophore or luminescent moiety, m is an integer from1 to 10, R¹ is NH, O, S or SO₂, R² is a hydrogen, a halogen, anisothiocyano group (SNC), an azido group (N₃), a sulfonate group(SO₃R⁴), a sulfate group (OSO₃R⁴), a carboxyl group (CO₂H), a carbonylgroup (COR⁴), an amido group (CONR⁴ ₂ or NR³COR⁴), a carbamate group(NR⁴CO₂R⁴), a phosphate group (OPO₃R⁴ ₃), a phosphonate group (PO₃R⁴ ₂),an amino group (NR⁴ ₂), an alkoxy group (OR⁴), a thiol group (SR⁴), asulfoxy group (SOR⁴), a sulfone group (SO₂R⁴), a sulfonamide group(SO₂NR⁴ ₂), a phosphino group (PR⁴ ₂), a silane group (SiR⁴ ₃), anoligopeptide sequence of 1-20 modified or unmodified amino acids oramino acid substitutes, a protein, a glycoprotein or a lipoprotein, eachR⁴ is independently a hydrogen, 1 to 3 halogen atoms, a substituted orunsubstituted C₁-C₁₀ straight-chain, branched or cyclic alkyl, alkenylor alkynyl group wherein one or more C, CH or CH₂ groups may besubstituted with an O atom, N atom, S atom, or NH group, anunsubstituted or substituted aromatic group wherein one or more C, CH orCH₂ groups may be substituted with an O atom, N atom, S atom, or NHgroup, and R³ is a methyl succinyl moiety; (b) incubating theenzyme-molecule-compound mixture to allow the enzyme to remove themethyl succinyl moiety in the absence of the molecule; and (c)determining whether the methyl succinyl moiety is removed from thecompound to an equivalent degree that the methyl succinyl moiety wouldbe removed from the compound in the absence of the molecule, wherein thefailure of the removal of the methyl succinyl moiety from the compoundto an equivalent degree as in the absence of the molecule indicates thatthe molecule is an inhibitor of the enzyme.
 2. The method of claim 1,wherein the enzyme is a histone deacetylase (HDAC).
 3. The method ofclaim 1, wherein the compound is a substrate for a peptidase after theenzyme cleaves the methyl succinyl moiety from the compound but not ifthe methyl succinyl moiety is not removed from the compound; wherein thedetermining step further comprises (i) adding the peptidase to themixture to allow the peptidase to cleave the resulting molecule betweenthe nitrogen bound to SIG and the carbonyl carbon bound to saidnitrogen, such that SIG generates an increased signal relative to thesignal generated with the compound; and (ii) determining whether SIGgenerates an increased signal relative to the signal generated with thecompound, wherein an increased signal indicates the removal of themethyl succinyl moiety from the compound.
 4. The method of claim 3,wherein the peptidase is a trypsin.
 5. The method of claim 3, whereinthe enzyme is a histone deacetylase (HDAC).
 6. The method of claim 5,wherein the peptidase is a trypsin.
 7. The method of claim 1, whereinSIG is a fluorophore.
 8. The method of claim 1, wherein SIG is aluminescent moiety.
 9. The method of claim 2, wherein SIG is afluorophore.
 10. The method of claim 2, wherein SIG is a luminescentmoiety.
 11. The method of claim 3, wherein SIG is a fluorophore.
 12. Themethod of claim 3, wherein SIG is a luminescent moiety.
 13. The methodof claim 1, wherein the compound is

wherein m is an integer from 1 to
 10. 14. The method of claim 13,wherein m=1.
 15. The method of claim 13, wherein m is an integer from 2to
 10. 16. The method of claim 1, wherein a plurality of differentmolecules are subjected to the method simultaneously.
 17. The method ofclaim 2, wherein a plurality of different molecules are subjected to themethod simultaneously.
 18. The method of claim 3, wherein a plurality ofdifferent molecules are subjected to the method simultaneously.